INTRODUCTION

HERBAL MEDICINE
Herbal medicines originated in ancient societies, and plants are used
medicinally to alleviate illness and enhance health and wellness. Also known as
herbalism or botanical medicine, it involves the consumption or topical application
of plant extracts. Herbalism has long been used outside conventional medicine. The
use of herbal medicines is becoming more mainstream as improvements in analysis
and quality control, along with advances in clinical research, demonstrate the value
of herbal medicines in treating and preventing diseases. Ever since the birth of
mankind, there has been a relationship between life, disease, and plants.

Herbal medicine is the oldest form of medicine still widely practiced today,
deriving its components entirely from plant parts such as leaves, roots, stems,
flowers and seeds. Herbal medicine is the study of pharmacognosy and the use of
medicinal plants, which are a basis of traditional medicine. The definition,
‘traditional’ use of herbal medicines implies substantial historical use, and this is
certainly true for many products that are available as ‘traditional herbal medicines. In
many developing countries, a large proportion of the population relies on traditional
practitioners and their armamentarium of medicinal plants in order to meet health
care needs.
A considerable number of definitions have been proposed for medicinal
plants. According to the WHO, “A medicinal plants is any plant which, in one on
more of its organs, contains substance that can be used for therapeutic purpose, or
which are precursors for chemo-pharmaceutical semi-synthesis.” it is implied that the
said plant is used as a drug or therapeutic agent or an active ingredient of a medicinal
preparation.
“Medicinal plants may therefore be defined as a group of plants that possess
some special properties that quality them as articles of drugs and therapeutic agents,
and are used for medicinal purposes”. Nature is a best friend of our pharmacy field.
Natural dugs are effective in action without side effects.

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 Natural products, including medicinal plants, have been the primary source
for obtaining new drugs with therapeutic potential throughout history. It is estimated
that approximately half of the drugs in use are derived from natural products. This
means that around 4 billion people rely on natural products as a source of their
primary medicinal needs. It is proved that half of the world’s best selling drugs and
many potential drugs under development are derived from plants.
Medicinal plants represent an important health and economic component of
biodiversity. Plants are the natural reservoir of many antimicrobial, anticancer
agents, analgesics, anti-diarrheal as well as various therapeutics activities. A lot of
medicinal plants are available for the treatment of various diseases. Natural products
and related drugs are used to treat 87% of all categorized human disease including
bacterial infection, cancer and immunological disorders. A large number of plants in
different location around the world have been extracted and semi-purified to
investigate individually their antimicrobial activity.
Medicinal plants are cheap and easy to get to those people who knew it very
well. Bioactive compounds are deposited in medicinal plants; it can serve as
important raw materials for pharmaceutical manufacturing.

Benefits of Herbal Medicine

 More affordable than conventional medicine.

 Easier to obtain than prescription medicine.
 Stabilizes hormones and metabolism.
 Natural healing.
 Strength in immune system.
 Fewer side effects.
 Cost effective.

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History of traditional medicine

The history of herbalism is closely tied with the history of medicine from
prehistoric times up until the development of the germ theory of disease in the 19th
century. Modern medicine from the 19th century to today has been based on
evidence gathered using the scientific method. Evidence-based use of pharmaceutical
drugs, often derived from medicinal plants, has largely replaced herbal treatments in
modern health care. However, many people continue to employ various forms of
traditional or alternative medicine. These systems often have a significant herbal
component. The history of herbalism also overlaps with food history, as many of the
herbs and spices historically used by humans to season food yield useful medicinal
compounds and use of spices with antimicrobial activity in cooking is part of an
ancient response to the threat of food-borne pathogens.

The germ theory of disease in the 19th century led to cures for many infectious
diseases. Military doctors advanced the methods of trauma treatment and surgery.
Public health measures were developed especially in the 19th century as the rapid
growth of cities required systematic sanitary measures. Advanced research centers
opened in the early 20th century, often connected with major hospitals. The mid-20th
century was characterized by new biological treatments, such as antibiotics. These
advancements, along with developments in chemistry genetics, and radiography led
to modern medicine. Medicine was heavily professionalized in the 20th century, and
new careers opened to women as nurses (from the 1870s) and as physicians
(especially after 1970).

Plants had been used for medicinal purposes long before recorded history.
Ancient Chinese and Egyptian papyrus writings describe medicinal uses for plants as
early as 3,000 BC. Indigenous cultures (such as African and American) used herbs in
their healing rituals, while others developed traditional medical systems (such as
Ayurveda and Traditional Chinese Medicine) in which herbal therapies were used.
Researchers found that people in different parts of the world tended to use the same
or similar plants for the same purposes.
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 In the early 19th century, when chemical analysis first became available,
scientists began to extract and modify the active ingredients from plants. Later,
chemists began their own version of plant compounds and, overtime, the use of
herbal medicine in favor of drugs. Almost one fourth of Pharmaceutical drugs are
derived from botanicals.

Recently, the World Health Organization estimated that 80% of people

worldwide rely on herbal medicines for part of their primary health care.

TYPES OF HERBAL MEDICINE

Ayurveda

Ayurveda - ancient science of life is believed to the top prevalent for last five
thousand years in India. It is one of most noted systems of medicine in the world.
Ayurveda based on the hypothesis that everything in the universe is composed of five
basic elements.

Space, air, energy, liquid and solid that exist in human body in combined forms like
vata, pitta, and kappa together are called tridosa.

Siddha

The term siddha means achievement and siddhars were saintly personalities who
proficiency in medicine through practice of bhakti and yoga. This is a system
identified with Dravidian culture and it largely therapeutic in nature.

Homeopathy

In comparison to other traditional system of medicine homeopathy is a newer

one and has been developed in the eighteenth century by Samuel Hahnemann -a
German physician and chemist to he proposed that the cause of disease itself can be
used for its treatment.

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Unani

The roots of this system go deep to times of the well known Greek Philosopher
Hippocrates who is credited with it. This system is based on two theories viz. the
Hippocratic theory four humours and Pythagorian theory of four qualities. The four
humours are blood, phlegm, yellow bile and black bile. while the four qualities are
the state of living human body like hot, cold, moist and dry. They are represented as
earth, water, fire and air. This system of medicine is otherwise called as Arab
medicine, Loniah medicine and Islamic medicine.

Herbal drugs

Herbal medicine also called botanical medicine or phytomedicine refers to the

use of plants seeds, berries, roots, leaves, bark or flowers for medicinal purposes. It
is used in all societies and is common to all culture.

Yoga

Yoga therapy is a type of therapy that uses yoga postures, breathing exercises,
meditation, and guided imagery to improve mental and physical health. The holistic
focus of yoga therapy encourages the integration of mind, body, and spirit.

 The world Health Organization (WHO) estimates that about 80% of the
population living in the developing countries rely almost exclusively on
traditional medicine for their primary health care needs. In almost all the
traditional medicine, the medicinal plants play a major role and constitute
the backbone of the traditional medicine. Indian material medical
includes about 2000 drugs of natural origin almost all of which are
derived from different traditional system and folklore practices. The
medicinal value of plants lies in some chemical substances or group of
compounds that produce a definite physiological action in the human
body. These chemical substances are called secondary metabolites.

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 The most important of these bioactive groups of plants are alkaloids,
terpenoids, steroids, flavonoids, tannins and phenolic compounds.
Infectious diseases caused by bacteria, fungi, viruses and parasites are
still a major threat to public health, despite the tremendous progress in
human medicine. Their impact is particularly large in developing countries
due to the relative unavailability of medicines and the emergence of
widespread drug resistance. Among the infectious diseases, fungal
infections account for a larger proportion of health problems in human
beings, particularly among women and children. The substances that can
inhibit pathogens and have little toxicity to host cells could be
considered candidates for developing new antimicrobial drugs . These
compounds are found in various medicinal plant parts such as stems, roots,
leaves, barks, flowers, fruits and seeds.

Decalepis hamiltonii is endemic endangered medicinal plant species and

It prefers to grow along rocky slopes, big rock boulders and rocky crevices
and small mounds where there is thick vegetation at an altitude from 300 to
1200m. It has good medicinal importance and used in wide drug preparations.
Pharmacognostical study of roots of D. hamiltonii was investigated for proper
identification during drug preparation. The pharmacognosy, phytochemistry and
pharmacology of D. hamiltonii were reviewed.

Plumbago zeylanica is a herbaceous plant grows throughout the tropical

and sub-tropical climates of the world including Australia and India. In Australia,
it grows in the understory of monsoon forests and vine thickets from sea level to
900 m. The aim of the present study is to study the antimicrobial activity of
[Link] medicinal plant.

 Plants are the important source of various useful materials and provide food,
shelter, medicine etc. Detailed descriptions of plant and plant’s products
about 700 herbs used for medicinal purposes to cure various ailments have

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 been mentioned in various literatures like Ayurveda, Charak samhita and
Susrut Samhita.
P. zeylanica is a medicinal plant which commonly known as “White
leadwort” or “Chitrak”. It belongs to the Plumbaginaceae family and a
perennial herb which is found in Uttar Pradesh, West Bengal, Maharashtra
and also to some parts of South India . It is one of the oldest herbs which are
reported to be used in Ayurveda for several disorders over thousands of years.
It contains various bioactive compounds like alkaloids, flavonoids,
napthoquinones, glycoside, saponins, steroids, tri-terpenoids, coumarins,
phenolic compounds, tannins, carbohydrate, fixed oils, fats and proteins. This
plant have been reported to show anti-bacterial, anti-plasmodial, anti-tumour,
hepatoprotective, central nervous system stimulatory activity, anti-fungal,
anti-inflammatory, anti-hyperglycemic, anti-cancer, antiatherosclerotic
activity etc. Leaves of P. zeylanica are dark green in colour and are simple,
elliptical with hairy margins along with alternate placement on the stem with
the distance of up to 3 inches and thickness of 1.5 inches. Petioles are thin
and with an approximate length of 0.5 mm and native stipules are present .
Plants breed flower white in colour with diameter of 1/2 to 3/4 inch having
the stalk measuring 4 to 12 inches along with a terminal raceme-type of
inflorescence. Roots of P. zeylanica are long and slightly branched with very
less secondary roots, having a smooth and unbroken texture, colour of the
roots is light yellow when the plant is freshly plucked out of the ground and
changes to reddish brown in colour when it is dried which often initiates in
the form of hard pieces. These roots are usually very strong having a bitter
taste and a distinct odour with acrid.

Today, herbal medicine serves as a bridge between ancient wisdom and modern
science, offering a natural approach to health while emphasizing the importance of
sustainable and informed use.

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LITERATURE REVIEW

 In February 2005, Antimicrobial Screening of Decalepis hamiltonii Wight and

Arn. (asclepiadaceae) Root Extracts against Food-related microorganisms by D.
Thangadurai, K.S.R. Murthy, P.J.N. Prasad, T. pullaiah.
The antimicrobial activity of petroleum ether, benzene, chloroform, ethyl acetate and
methanol extracts of Decalepis hamiltonii Wight and Arn. (Asclepiadaceae) against
15 different food-related microorganisms was evaluated. Among the solvent extracts,
methanolic and petroleum ether extracts are the most. The inhibitory activity was
comparable with the respective standards. The results suggest that methanolic and
petroleum ether extracts can be used for the bioactivity-guided isolation of novel
antimicrobials which prevents food spoilage.

 In June 2008, Antibacterial Activity Of Plumbagin and Root Extracts of

Plumbago zeylanica by L.R. Jeyachandran, AMahesh, L. Cindrella, S. Sudhakar,
and K. Pazhanichamy.
This work assess the Anti-bacterial activity of plumbagin (5-hydroxy-2-
methylnaphthalene-1,4-dione) and of methanol, chloroform and aqueous extracts of
Plumbago zeylanica L. root against various pathogenic bacteria,and the minimum
inhibitory concentrations (MICs). Plumbagin and chloroform extracts of Plumbago
zeylani-ca L. root showed antibacterial activity against Escherichia coli, Salmonella
typhi and Staphylococcus aureus. Inhibition against Klebsiella pneumoniae, Serratia
marcescens and Bacillus subtilis was moderate, and lower against Proteus vulgaris
and Pseudomonas aeruginosa. The methanolic extract exhibited moderate activity
and the aqueous extract weak activity against the bacterial strains as assessed by disc
diffusion assays. The bioactive compound plumbagin and extract of Plumbago
zeylanica root show a wide spectrum of antibacterial activity. The compound shows
promise as a new drug for various bacterial infectious diseases.

 In December 2009, Insecticidal activity of the root extract of Decalepis

hamiltonii against stored-product insect pests and its application in grain
protection by Y. Rajashekar, Nivedhitha Gunasekaran, T. Shivanandappa.
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The root extracts of Decalepis hamiltonii were tested for insecticidal activity against
the stored products pests, Rhyzopertha domonica, Sitophilus oryzae, Stigobium
pancieum, Tribolium castaneum and Callosobruchus chinensis, in residual and
contact toxicity bioassays. Methanolic extract showed LC50 value of 0.14 mg/cm2
for all the test species in a fi lter paper residual bioassay. The extract was effective as
a grain protectant for wheat and green gram. Reduction of F1 progeny was observed
in treated grain stored for 3–4 months. The extract did not affect the germination of
the treated grains. Our results indicate that methanolic extracts of D. hamiltonii has a
potential to control stored product pests and could serve as a natural grain protectant.

 In November 2014, Antimicrobial activity of Plumbago zeylanica plant extracts

and its application in water and laboratory disinfection Ayoade A. Adegbitea,
Abdulazeez A, Adebanjob , A.M YusufBabatundec , Obafemi [Link] and
Risikat A. Sowolee.
This study was carried out to investigate the antimicrobial potentials of Plumbago
zeylanica components (leaf, stem and root) on four bacterial species, Baccilus
subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia with the
aim of using the active part of the plant to be used in water and laboratory
disinfection. The plant parts crude extract was concentrated using a rotary evaporator
and dried in a freeze drier. Different concentrations of the plant parts were then
prepared from the dried plant extract and tested on the four pathogens using agar
diffusion methods. The results indicated that active antimicrobial properties are
concentrated more in the roots been very effective against Escherichia coli even at
low concentration. The study concludes that the roots of Plumbago zeylanica possess
the highest antimicrobial potentials for disinfection. Then, the root extract was tested
on effluent water and the results showed significance reduction level of Escherichia
coli.

 In August 2016, Antimicribial Activity of Methanolic Root Extract of Decalepis

hamiltonni WIght & Arn by Prakash P., Manivasagaperumal.

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This study evaluated the antimicrobial activity of Decalepis hamiltonii root extracts
(petroleum ether, chloroform, ethyl acetate, and methanol) against gram-positive
bacteria (Bacillus subtilis, Streptococcus pyogenes), gram-negative bacteria, and
fungi (Candida albicans) using the disc diffusion method (250–1000 µL). Methanolic
extracts showed the highest inhibition zones, correlating with higher phenolic and
flavonoid content. These findings highlight the extract's potential for pharmaceutical,
therapeutic, and food preservation applications.

 In July 2016, Antimicrobial Activity of Methanolic Root Extract of Decalepis

hamiltonii Wight & Arn by P. Prakash and R. Manivasagaperumal.
This study was quantitative analysis and antimicrobial activity of petroleum ether,
chloroform, ethyl acetate, methanol root extracts. The gram positive and gram
negative bacterial and fungal species by the disc diffusion method at various
concentration (250µl-1000µl). All the root extract against to Bacillus subtilis and
Streptococcus pyogenes and fungal species Candida albicans at highest inhibition at
concentration of methanolic root extract of zone formation. The preliminary studies
on Decalepis hamiltonii root extract exhibited their antimicrobial potential

 In February 2017, Phytochemical screenig and Antimicrobial activity of root

extracts of decalepis hamiltonni Wight & Arn by Prakash P., G. Thiyagarajan,
Rengarajan Manivasagaperumal.
This study evaluated the phytochemical and antibacterial activity of petroleum ether,
chloroform, ethyl acetate, and methanol root extracts of Decalepis hamiltonii.
Phytochemical analysis revealed the presence of glycosides, steroids, flavonoids,
phenols, terpenoids, saponins, and tannins. Antibacterial activity, tested using the
disc diffusion method against seven bacterial pathogens, showed methanol extract to
be the most effective, particularly at higher concentrations. Klebsiella pneumoniae
was the most resistant, while Staphylococcus aureus, Bacillus cereus, and Bacillus
subtilis were least resistant. The findings support the traditional use of D. hamiltonii
and highlight its potential for developing antimicrobial agents.

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 In May 2019, In vitro antibacterial activity of plumbagin isolated from
Plumbago zeylanica L. against methicillin-resistant Staphylococcus aureus by H.
Periasamy,S. Iswarya, N. Pavithra, S. Senthilnathan, A. Gnanamani.
Plumbagin, a bioactive compound isolated from Plumbago zeylanica L., was
evaluated for its antimicrobial activity against methicillin-resistant Staphylococcus
aureus (MRSA), including multi-drug-resistant (MDR) strains. It demonstrated
potent activity with a narrow MIC range (4–8 μg/ml) against 100 MRSA isolates. A
time-kill assay revealed a 99% reduction in MRSA viability within 8 hours of
exposure. Combination MIC studies showed synergistic effects with ciprofloxacin
and piperacillin, while interactions with other antibiotics were either additive or
indifferent. Transmission electron microscopy confirmed plumbagin-induced
structural damage to the MRSA cell wall and cytoplasm. These findings suggest
plumbagin as a potential therapeutic agent against MRSA, warranting further
research for clinical applications.

 In 2021, Decalepis hamiltonii (Swallow Root) as a Potential Antimicrobial

Agent against Endodontic Pathogens: An In Vitro Study by Anand V Susila,
Selvam Sangeeta
The aim of the study was to evaluate and compare the antimicrobial effect of
alcoholic and hydroalcoholic extracts of Decalepis hamiltonii with those of Curcuma
longa, Azadirachta indica, and Zingiber officinale against Enterococcus faecalis.
Alcoholic and hydroalcoholic extracts of the herbs were prepared by cold maceration
and filtration–decantation process. Minimum bactericidal concentration (MBC) and
minimum inhibitory concentration (MIC) were determined for each extract; zone of
inhibition (ZOI) was assessed separately for each extract and their different
combinations on both laboratory strain and clinical isolates of E. faecalis. The
extracts of D. hamiltonii showed a significant antimicrobial action, against
endodontic pathogen E. faecalis, both independently and in combination
preparations. It concludes that the Extract of D. hamiltonii can be used as intracanal
irrigant and medicament in endodontics after confirming its biocompatibility.

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 In August 2023, Antimicrobial, antioxidant, anticancer, and antithrombotic,
competency of saponins from the root of Decalepis hamiltonii by Jayaraman
Gitanjali, Duraisamy Sumathi Dinesh Ram , Kavitha R, Venkatesan Amalan,
Tahani Awad Alahmadi, Sulaiman Ali Alharbi, Sabariswaran Kandasamy,
Rajasree Shanmuganthan, Natesan Vijayakumar.
The goal of this study was to extract saponins from the tuberous root of Decalepis
hamiltonii and assess their potential clinical applications, which included
antioxidant, antibacterial, antithrombotic, and anticancer properties. Surprisingly, the
results of this study revealed that the extracted saponins have excellent antioxidant
activities, as demonstrated by 2,2-diphenylpicrylhydrazyl (DPPH), 2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS), Hydrogen peroxide (H2O2), and
Nitric oxide (NO) scavenging assays. Nonetheless, at a concentration of 100 g/mL,
crude saponin had excellent antibacterial activity, particularly against gramme
positive bacteria (Staphylococcus aureus, Bacillus subtilis, Staphylococcus
epidermidis, and Micrococcus luteus), followed by gram negative bacteria
(Escherichia coli, Salmonella typhi, Proteus mirabilis, and Klebsiella pneumonia).
Despite this, the crude saponin had no effect on Aspergillus niger and Candida
albicans. The crude saponin also possesses outstanding in vitro antithrombotic
activity on blood clot. Interestingly, the crude saponins have an outstanding
anticancer activity of 89.26%, with an IC50 value of 58.41 μg/mL. Overall, the
findings conclude that crude saponin derived from D. hamiltonii tuberous root could
be used in pharmaceutical formulations.

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AIM AND OBJECTIVES.

Aim
To evaluate the antimicrobial activity of ethanolic root extracts of Plumbago
zeylanica and Decalepis hamiltonii by integrating advanced phytochemical analysis
techniques, including IR, and UV to identify bioactive compounds and assess their
antimicrobial efficacy.

Objectives

1. Extraction and Preparation of Plant Extracts

 To prepare ethanolic root extracts of Plumbago zeylanica and Decalepis
hamiltonii using optimized extraction protocols
2. Phytochemical and Spectroscopic Analysis
 To perform infrared (IR) spectroscopy to identify functional groups present in
the extracts
 To conduct UV spectroscopy to determine the absorbance and identify
chromophores related to bioactive compounds
3. Evaluation of Antimicrobial Activity
 To assess the antimicrobial activity of the extracts against bacterial and fungal
pathogens using disc diffusion method
4. Comparative and Correlation Analysis
 To compare the antimicrobial efficacy of Plumbago zeylanica and Decalepis
hamiltonii.
 To correlate the phytochemical composition (IR, UV, and HPTLC results) with
observed antimicrobial activity
5. Potential Applications and Future Directions
 To evaluate the potential of the extracts as natural antimicrobial agents and
provide recommendations for further development into pharmaceutical
applications

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PLAN OF WORK

Plant Selection & Collection

Authentication

Literature Review

Pulverisation of the root

Extraction of Plant materials

Macroscopical Investigation

Chromatographic
Preliminary Phytochemical Screening studies &
Phytochemical
Spectral Analysis
Screening
Preformulation Studies

Formulation Development

Evaluation Studies

Antimicrobial activity

Result & Discussion

Summary & Conclusion

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PLANT PROFILE.

 PLUMBAGO ZEYLANICA

Plumbago zeylanica, commonly known as white leadwort, is a medicinal plant

extensively used in traditional medicine systems like Ayurveda, Siddha, and Unani. It
belongs to the family Plumbaginaceae. This plant is renowned for its significant
pharmacological properties, including antimicrobial, anti-inflammatory, and anticancer
activities, which are attributed to its rich phytochemical composition. The roots are
rich in plumbagin and form the principal part used in traditional remedies, although
leaves and stems are also occasionally utilized in certain regional practices.

Synonyms : Plumbago scandens, Plumbago rosea, Plumbago zeylanica var. repens

Common Names
English : White Plumbago, Ceylon Leadwort
Tamil : Chitiramoolam, Kodivaeli
Hindi : Chitrak
Kannada : Chitramula
Telugu : Tella chitramulamu
Malayalam : Kottuveli
Oriya : Ogni
Bengali : Safaid - sitarak

GEOGRAPHICAL DISTRIBUTION

It is widely distributed across tropical and subtropical regions of the world. Globaly it
is Present in Africa, Sri Lanka, China, and Southeast Asia. In India, it is found in
forests and along the roadsides in states like Tamil Nadu, Kerala, and Maharashtra.

Plumbago zeylanica is widely adapted to a variety of environments, It prefers sandy or

loamy soils with adequate drainage. Commonly grows in tropical climates,
particularly in subtropical forests and semi-arid zones.
Found in open spaces, often near water bodies or cultivated fields. In India, it is seen
in the Western Ghats, Eastern Ghats, and Himalayan foothills.

TAXONOMICAL CLASSIFICATION

Kingdom : Plantae
Subkingdom : Tracheobionta
Superdivision : Spermatophyta
Division : Magnoliphyta
Class : Magnoliopsida
Subclass : Caryophyllidae
Order : Caryophyllales
Family : Plumbaginaceae
Genus : Plumbago
Species : Plumbago zeylanica
MORPHOLOGY
Plumbago zeylanica is a perennial herbaceous plant or climber with trailing or
ascending stems. It exhibits distinct morphological features:
 Stem: Woody at the base, herbaceous towards the apex, and slender.
 Leaves: Broadly ovate with a blunt tip, often exhibiting a slightly hairy
surface.
 Flowers: The inflorescence is a spiked raceme, with each flower featuring a
funnel-shaped calyx and five petals.
 Fruit: Small, capsular, containing a single seed.
The plant exudes a resinous secretion that is sticky and often acts as a defense
mechanism against herbivores.
 Fig. No: 1 Fruit, Flower, Leaves, & Root of the plant Plumbago zeylanica.

CHEMICAL CONSTITUENTS AND MEDICINAL PROPERTIES

P. zeylanica contains a variety of important chemical compounds. Different plant parts

of the plant possess naphthaquinones, alkaloids, glycosides, steroids, triterpenoids,
tannins, phenolic compounds, flavanoids, saponins, coumarins, carbohydrates, fixed
oil and fats and proteins. Of all the chemical constituents’ plumbagin is the principle
active compound. Plumbagin (5-hydroxy-2-methyl-1, 4- naphthoquinone- C 11H8O3) is
primarily present in roots in higher amounts with only about 1% in the whole plant. A
wide range of medicinal properties of P. zeylanica are attributed to Plumbagin and
other secondary metabolites. Plumbagin has shown antibacterial activity against both
gram-positive (e.g. Staphylococcus, Streptococcus, Pneumonococcus sp.) and gram
negative (e.g. Salmonella, Neisseria) bacteria. It is also active against certain yeasts
and fungi (Candida, Trichophyton, Epidermophyton and Microsporum spp.) and
protozoa (Leishmania).

Constituents & Activity

 Plumbagin : Anti microbial, anticancer, anti-

inflamatory, antioxidant
 3 - Chloroplumbagin : Antimicrobial, antitumour activity
  Steroids & Terpenoids : Anti inflammatory, antimicrobial,
Immunomodulatory.
 Alkaloids : Sedative, analgesic,antimicrobial,
anticancer
 (indole based)
 Napthoquinones : Antimicrobial, antimalarial, anticancer
properties
 Glycosides : Laxative, antimicrobial, anti oxidant
( Anthraquinone glycoside)
 Flavanoids (catechin) : Antioxidant, anti-inflammatory,
antimicrobial
 Tannins : Astrigent, anti diarrheal, antimicrobial
properties
 Essential Oils : Anti microbial, Anti inflammatory,
anti spasmodic

MEDICINAL USES

The root of the Plumbago zeylanica plant, has many uses in traditional medicine:

 Hepatoprotective: The root has potent hepatoprotective properties.

 Anti-inflammatory: The root has anti-inflammatory properties.
 Anti-hyperlipidemic: The root has anti-hyperlipidemic properties.
 Laxative: The root has laxative properties.
 Stomachic: The root has stomachic properties.
 Tonic: The root has tonic properties.
 Appetizer: The root has appetizer properties.
 Stimulates central nervous system: The root stimulates the central nervous system.
 Rheumatism, joint pain, and paralysis: Oil prepared from roots is useful in
rheumatism, joint pain, and paralysis.
  DECALEPIS HAMILTONII

Decalepis hamiltonii, an endemic plant of peninsular India, is a valued species

known for its versatile applications in traditional medicine, food flavoring, and
aromatherapy. The plant’s roots are consumed raw or as a syrup in rural areas for
improving vitality and protecting against infections. Recent pharmacological studies
highlight its antioxidant, antimicrobial, and hepatoprotective properties, making it a
promising candidate for natural product research.

Common Names
English : Indian Sarsaparilla
Tamil : Makali Kizhang
Hindi : Ananta
Kannada : Mahali
Telugu : Maredu Kommulu
Malayalam : Nannari
Sanskrit : Sugandhi

GEOGRAPHICAL DISTRIBUTION
Decalepis hamiltonii, commonly known as Swallow Root, is a climbing shrub
of great medicinal and aromatic value, primarily found in India. This species thrives in
various habitats, In India it found abundantly in Karnataka, Tamil Nadu, Andhra
Pradesh, and Kerala. It Grows in dry deciduous forests, shrublands, and rocky terrains
at altitudes up to 1200 meters. Conservation efforts are underway in regions like the
Eastern Ghats and Western Ghats.

MORPHOLOGY

 Roots: Highly aromatic, slender, and tuberous with longitudinal ridges; exudes
a vanilla-like odor due to 2-Hydroxy-4-methoxybenzaldehyde. The roots also
secrete a sticky resinous substance.
  Stem: Twining, terete, and moderately woody; green when young, turning
brown with age.
 Leaves: Thick, leathery, and oblong with parallel venation; glossy on the upper
surface and pale green beneath.
 Flowers: Pentamerous, small, and tubular with a light yellow or greenish hue.
The corolla is campanulate (bell-shaped).
 Fruit: Narrow follicles (2.5–3 cm long), each containing numerous seeds with
silky hairs aiding wind dispersal.

[Link].2 : Different plant parts of D. hamiltonii

A-Cylindrical tuberous roots excised from a five year old plant.

B-Dry follicle with seeds.
C-Seeds with silky hairs.
D-Fresh seeds.
E-Complete plant of D. hamiltonii planted in loamy soil-6 month old.
TAXONOMICAL CLASSIFICATION

Kingdom : Plantae
Phylum : Tracheophyta
Class : Mangoliopsida
Order : Gentianales
Family : Apocynaceae
Subfamily : Periplocoideae
Genus : Decalepis
Species : Decalepis hamiltonii Wight & Arn

CHEMICAL CONSTITUENTS AND MEDICINAL PROPERTIES

Decalepis hamiltonii, commonly known as Swallow Root, is a medicinal plant with

notable therapeutic and aromatic properties. It is rich in bioactive compounds like 2-
hydroxy-4-methoxybenzaldehyde, which exhibits strong antimicrobial, antioxidant,
and anti-inflammatory activities. The plant contains flavonoids, triterpenoids, and
saponins, enhancing its pharmacological profile. Traditionally, it is used to manage
digestive issues, respiratory ailments, and inflammation. Its antibacterial spectrum is
particularly significant, showing activity against pathogens such as Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. These
properties make Decalepis hamiltonii a promising candidate for treating bacterial
infections, especially in an era of rising antibiotic resistance.

Constituents & Activity

2 - Hydroxy-4-Methoxy : Antimicrobial, antioxidant, anti inflammatory
Vanillin : Antioxidant, anti inflammatory, Flavoring agent
Coumarins : Antioxidant, anti coagulant,anti inflammatory
Flavanoids(Kaempferol, : Antioxidant, cardioprotective, anti inflammatory
Quercitin, Rutin)
Alkaloids(Reserpine) : Hypotensive, anti microbial agent
Saponins(Diosgenin) : Anti inflamatory, wound healing activity
 Terpinoids(Lupenol ) : Anti inflamatory, cholesterol lowering,
Hepatoprotective

MEDICINAL USES

Decalepis hamiltonii is used for variety of purposes:

 Blood purifier: Decalepis hamiltonii is used as a blood purifier

 Appetizer: Decalepis hamiltonii is used to stimulate the appetite
 Wound healer: Decalepis hamiltonii is used to heal wounds
 Fever: Decalepis hamiltonii is used to treat fever
 Inflammation: Decalepis hamiltonii is used to reduce inflammation
 Bronchial asthma: Decalepis hamiltonii is used to treat bronchial asthma
 Digestive problems: Decalepis hamiltonii is used to treat digestive problems
 Skin diseases: Decalepis hamiltonii is used to treat skin diseases
 Food preservative: Decalepis hamiltonii is used as a food preservative due to its
antioxidant potential
MATERIALS AND METHODS

Phase 1: Literature Review and Planning

 Conduct a thorough literature review on Decalepis hamiltonii and Plumbago

zeylanica, focusing on their phytochemistry, pharmacology, and traditional
uses.
 Identify the research gap and formulate a research question.
 Develop a detailed research plan, including objectives, methodology, and
timelines.

Phase 2: Plant Material Collection and Extraction

 Collect the roots of Decalepis hamiltonii and Plumbago zeylanica from

authenticated sources.
 Clean, dry, and powder the plant material.
 Extract the phytoconstituents using a suitable solvent (e.g., ethanol)

Phase 3: Formulation Development

 Develop a cream formulation using the extracted phytoconstituents.

 Optimize the formulation using different polymers, solvents, and additives.
 Evaluate the physical characteristics (e.g., texture, viscosity, pH) of the
formulated cream.

Phase 4: Analytical Studies

 Perform UV-Visible spectroscopy to identify the phytoconstituents.

 Conduct IR spectroscopy to determine the functional groups present.

Phase 5: Evaluate the synergistic antimicrobial potential

 Evaluate the antimicrobial activity against multidrug-resistant bacteria (e.g., E.

coli, S. aureus) using in vitro (e.g., agar well diffusion, broth microdilution)
assays.
  Determine the minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) of the cream formulation.

Phase 6: Data Analysis and Interpretation

 Analyze the data from the analytical studies, pharmacological screening, and
antimicrobial study.
 Interpret the results in the context of the research question and objectives.

Phase7: Manuscript Preparation and Submission

 Prepare a manuscript based on the research findings.

 Edit and revise the manuscript according to the journal's guidelines.
 Submit the manuscript to a peer-reviewed journal.
7.1 EXTRACTION OF PLANT MATERIAL

Preparation of Ethanolic Root Extract from Plumbago zeylanica and Decalepis

hamiltonii

1. Collection and Authentication of Roots

The roots of Plumbago zeylanica and Decalepis hamiltonii were sourced from a
locally authenticated supplier to ensure the quality and identity of the plant materials.

2. Powdering of Roots

The collected roots were cleaned thoroughly to remove any adhering soil and debris.
They were dried under shade to retain their phytochemical integrity. The dried roots
were then finely powdered using a grinder and the powdered material was weighed
accurately.

3. Defatting of Root Powder

The powdered roots were subjected to defatting to remove lipophilic impurities.

Solvents Used: Ethyl acetate and chloroform.

The powdered root material was subjected to extraction using a Soxhlet apparatus in
the solvents sequentially to extract non-polar compounds. This step helps in enhancing
the extraction efficiency of the polar solvents used later.

4. Soxhlet Extraction with Ethanol

The defatted root powder was again subjected to extraction using the Soxhlet
apparatus.

Solvent Used: Ethanol.

Duration: The Soxhlet extraction was carried out continuously for 3 hours.

During this process, ethanol circulated through the Soxhlet apparatus, efficiently
extracting the active phytochemical constituents from the root powder.
Fig. No : 3 Extraction of Ethanolic Root Extract from [Link] using Soxhlet
Apparatus.
Fig. No : 4 Extraction of Ethanolic Root Extract from [Link] using Soxhlet
Apparatus.
 5. Separation of Ethanol

After completing the Soxhlet extraction, the ethanol extract was collected and
subjected to distillation to recover the ethanol.

Distillation: The ethanol was evaporated under reduced pressure using a distillation
setup to concentrate the extract.

Fig. No : 5 Distillation of Ethanolic Root Extract from Decalepis hamiltonii and

Plumbago zeylanica

6. Removal of Residual Ethanol

The concentrated extract was further heated on a hot plate at controlled temperatures
to ensure complete removal of residual ethanol. This step prevented any contamination
of the final extract with solvent residues.

7. Final Extract

After the complete evaporation of ethanol, a semi-solid ethanolic root extract of

Plumbago zeylanica and Decalepis hamiltonii was obtained.
7.2 PHYTOCHEMICAL SCREENING
 Phytochemical Screening of Decalepis hamiltoni Root Extract
The [Link] extract of Ethanolic root was subjected to preliminary
phytochemical screening to test for the presence or absence of phytochemical
constituents .

 Alkaloids

1 ml extract was stirred with 5 ml dil. Hydrochloric acid on a steam bath, filtered and
1ml of each Dragendoff’s reagents was added to 1ml separate portions of filtrate. A
cloudy Reddish brown color indicated the presence of alkaloids.

 Carbohydrate tests

1ml extract was added to 1ml Molisch’s reagent and 1ml conc. H 2SO4 was carefully
added. A violet ring indicates the presence of carbohydrates.

 Flavanoids (Shinoda test)

1ml extract was dissolved in ethanol, warmed and then filtered. Three pieces of
magnesium chips was then added to the filtrate followed by few drops of conc. HCl.
Pink Scarlet colouration indicates the presence of flavonoids.

 Cardiac glycosides (Keller- Kiliani test)

The drug was extracted with chloroform and evaporated to dryness. Then mixture of
glacial acetic acid (2 ml) was added with 2 drops of 2% ferric chloride. Carefully
0.5ml of concentrated sulphuric acid was added by the side of test tube. Acetic acid
layer shows blue colour and later on a brown ring was produced.

 Saponin glycosides (Froth formation test)

2ml solution of drug was added to water in a test tube, shake well, no stable froth
(foam) is formed.
  Proteins (Biuret Test)

To the drug extract, biuret reagent (2ml) was [Link] Violet colour is formed that
indicates the presence of protein.

 Tannins

1 ml extract was stirred with 1 ml Ferric chloride. A greenish black precipitate

indicates the presence of tannins.

 Terpenoids &Steroids (Salkowski’s test)

0.5 g extract was dissolved in 2.0 ml chloroform and 1.0 ml conc. H2SO4 was
carefully added. A reddish brown color indicates the presence of steroids.

Table No. 1 – Phytochemical Screening of the Ethanolic Extract of [Link].

[Link] CONSTITUENTS CHLOROFORM ETHYL ETHANOLIC

ETRACT ACETATE EXTRACT

EXTRACT
1. Alkaloids + + +
2. Carbohydrate + + +
3. Flavanoids + + +
4. Cardiac glycosides + + +
5. Saponin glycosides - + -
6. Proteins - - -
7 Tannins - + +
8. Terpenoids &Steroids + + +

(+) – Presence of Constituents

(-) – Absence of Constituents

  Phytochemical Screening of Plumbago zeylanica Root Extract.
The P,zeylanica extract of Ethanolic root was subjected to preliminary
phytochemical screening to test for the presence or absence of phytochemical
constituents .

 Alkaloids

1 ml extract was stirred with 5 ml dil. Hydrochloric acid on a steam bath, filtered and
1ml of each Dragendoff’s reagents was added to 1ml separate portions of filtrate. A
cloudy Reddish brown color indicated the presence of alkaloids.

 Carbohydrate tests

1ml extract was added to 1ml Molisch’s reagent and 1ml conc. H 2SO4 was carefully
added. A violet ring indicates the presence of carbohydrates.

 Flavanoids (Shinoda test)

1ml extract was dissolved in ethanol, warmed and then filtered. Three pieces of
magnesium chips was then added to the filtrate followed by few drops of conc. HCl.
Orange colouration indicates the presence of flavonoids.

 Cardiac glycosides (Keller- Kiliani test)

The drug was extracted with chloroform and evaporated to dryness. Then mixture of
glacial acetic acid (2 ml) was added with 2 drops of 2% ferric chloride. Transfer to a
small test tube; Carefully 0.5ml of concentrated sulphuric acid was added by the side
of test tube. Acetic acid layer shows blue colour and later on a brown ring was
produced between the layers which indicate the entity of cardiac steroidal glycosides.

 Saponin glycosides (Froth formation test)

2ml solution of drug was added to water in a test tube, shake well, stable froth (foam)
is formed.
  Tannins

1 ml extract was stirred with 1 ml Ferric chloride. A greenish black precipitate

indicates the presence of tannins.

 Terpenoids &Steroids (Salkowski’s test)

0.5 g extract was dissolved in 2.0 ml chloroform and 1.0 ml conc. H2SO4 was
carefully added. A reddish brown color indicates the presence of steroids.

Table No. 2 – Phytochemical Screening of the Ethanolic Extract of [Link].

[Link] CONSTITUENTS CHLOROFORM ETHYL ETHANOLIC

ETRACT ACETATE EXTRACT
EXTRACT
1. Alkaloids + + +
2. Carbohydrate + + +
3. Flavanoids + + +
4. Cardiac glycosides + + +
5. Saponin glycosides - - +
7 Tannins + + +
8. Terpenoids &Steroids + + +

(+) – Presence of Constituents

(-) – Absence of Constituents

DEVELOPMENT OF ANALYTICAL STUDIES

8.1 UV SPECTROSCOPY IN THE ANALYSIS OF ETHANOLIC ROOT

EXTRACTS OF PLUMBAGO ZEYLANICA AND DECALEPIS HAMILTONII.

UV-Visible Spectroscopy is a powerful and essential analytical technique. It provides

detailed insights into the molecular structure and composition of plant-derived
compounds by analyzing their interaction with ultraviolet light. In this study, the
ethanolic root extracts of Plumbago zeylanica and Decalepis hamiltonii are evaluated
using UV spectroscopy to identify and characterize bioactive compounds responsible
for their antimicrobial activity.

Principle of UV Spectroscopy

UV-Visible spectroscopy is based on the absorption of ultraviolet (UV) light by

molecules in the range of 200-400 nm, resulting in electronic transitions between
molecular orbitals. These transitions provide valuable information about the
chromophores and conjugated systems present in the sample.

Application of UV Spectroscopy in Plumbago zeylanica and Decalepis hamiltonii

The ethanolic root extracts of these plants are rich in secondary metabolites with
significant therapeutic potential:
1. Compounds in Plumbago zeylanica:
Plumbagin: A quinonoid compound known for its antimicrobial, antifungal, and
anticancer properties. Plumbagin has a characteristic UV absorption peak around 260-
280 nm (attributed to π → π* transitions in the aromatic ring) and another peak near
320-340 nm (due to its extended conjugation).
2. Compounds in Decalepis hamiltonii:
Decalepic Acid and Phenolics: These compounds exhibit antimicrobial and antioxidant
activity. They show prominent UV absorption between 270-300 nm, typical of
phenolic compounds.
Interpretation of the UV Spectrum of Decalepis Hamiltonni
1. Peak at 205.1 nm (Absorbance: 4.000 AU):
This strong absorbance in the UV-C region indicates the presence of π→π*
electronic transitions. These are typically associated with highly conjugated
systems or aromatic rings.
2. Peak at 220.5 nm (Absorbance: 0.606 AU):
This peak in the near UV-C region is likely due to π→π* transitions,
possibly from less conjugated chromophores or partially conjugated systems.
3. Peak at 279.9 nm (Absorbance: 0.208 AU):
This peak lies in the UV-B region and suggests n→π* transitions, which
are typically associated with non-bonding electrons (e.g., lone pairs) in carbonyl
groups or similar chromophores.
Conclusion:

The UV spectrum reveals distinct electronic transitions: π→π* at 205.1 nm and

220.5 nm, indicating conjugated or aromatic systems, and n→π* at 279.9 nm,
suggesting the presence of carbonyl or related functional groups. These results are
consistent with a compound containing conjugated systems with electron-donating or
withdrawing groups, as well as non-bonding electron-rich moieties.
Interpretation of the UV Spectrum Plumbago zeylanica
1. Peak at 210.0 nm (Absorbance: 4.000 AU):
This strong absorption in the UV-C region indicates a π→π* transition,
typically associated with highly conjugated or aromatic systems.
2. Peak at 220.6 nm (Absorbance: 3.968 AU):
This peak, also in the UV-C region, represents another π→π* transition, likely
due to a less conjugated system compared to the first peak.
3. Peak at 274.9 nm (Absorbance: 0.134 AU):
This peak in the UV-B region suggests an n→π* transition, commonly linked
to functional groups like carbonyls or other groups containing lone pairs of electrons.
Conclusion

The UV spectrum reveals prominent π→π* transitions at 210.0 nm and 220.6

nm, signifying the presence of conjugated or aromatic chromophores. Additionally, the
n→π* transition at 274.9 nm indicates the presence of carbonyl or similar electron-
donating functional groups. This suggests a compound with both conjugated systems
and lone-pair-containing groups.
8.2 INFRARED (IR) SPECTROSCOPY IN THE ANALYSIS OF ETHANOLIC
ROOT EXTRACTS OF PLUMBAGO ZEYLANICA AND DECALEPIS
HAMILTONII

Infrared (IR) Spectroscopy is a versatile analytical technique widely used for

identifying functional groups and studying molecular structures in natural products. It
is particularly useful for characterizing phytochemicals in plant extracts, such as the
ethanolic root extracts of Plumbago zeylanica and Decalepis hamiltonii, as it provides
a "fingerprint" of their chemical composition.

Principle of IR Spectroscopy

IR spectroscopy is based on the absorption of infrared radiation by molecules, which

causes vibrations in chemical bonds. When a molecule absorbs IR radiation, the
energy is converted into vibrational energy, resulting in characteristic absorption bands
that correspond to specific functional groups and molecular structures.

The absorption occurs in the mid-IR region, typically ranging from 4000 to 400 cm⁻¹,
and each functional group produces a unique absorption pattern, making it possible to
identify the chemical constituents.

Applications of IR Spectroscopy in Plumbago zeylanica and Decalepis hamiltonii

The ethanolic root extracts of these plants are rich in secondary metabolites with
diverse functional groups. IR spectroscopy helps in identifying these groups and
confirming the presence of bioactive compounds:

1. Compounds in Plumbago zeylanica

Plumbagin: A naphthoquinone derivative with functional groups such as carbonyl

(C=O), hydroxyl (-OH), and aromatic systems.

The carbonyl group exhibits a strong absorption band around 1650-1750 cm⁻¹, while
hydroxyl groups show broad bands around 3200-3600 cm⁻¹.
2. Compounds in Decalepis hamiltonii

Decalepic Acid: Contains phenolic (-OH), carbonyl (C=O), and possibly carboxylic
acid groups.

Phenolic groups exhibit absorption bands near 3200-3600 cm⁻¹, and carboxylic acid
groups show bands around 1700-1750 cm⁻¹.

By analyzing the IR spectrum, it is possible to confirm the presence of these functional

groups and gain insights into the chemical structures of bioactive compounds.
IR Interpretation of Decalepis hamiltonni

WAVE NUMBER
SIGNAL INTERPRETATION
(cm-1)

3949.82, 3854.16 Broad signals in this range indicate weak O-H stretching
vibrations (free OH groups)

3356.78 Likely corresponds to O-H stretching vibrations, indicative of

alcohol or phenol, carboxylic acid.

2918.86 C-H stretching vibrations of aliphatic (sp³-hybridized) groups,

suggesting the presence of alkane chains.

2099.20 Possible C=C stretching vibrations.

1447.49, 1607.45 These peaks typically represent C=C stretching vibrations in

Aromatic rings.

1488.56, 1399.39 -C-H bending vibrations in aromatic compounds or CH2/CH3

deformation modes.

1044.47 C-O stretching vibrations, indicating the presence of alcohols.

873.45, 822.54 Out-of-plane C-H bending vibration in aromatic compoundss

606.58 Indicates aromatic C-H out of plane bending.

441.93 Low-frequency signals are usually associated with skeletal

vibrations

Table No.3 – Interpretation of IR Spectrum of Decalepis hamiltonii.

Conclusion:

Based on the IR spectrum:

 Broad bands at 3356 cm-1 and 3854-3949 cm-1 suggest the presence of
hydroxyl (O-H) groups.
 2919 cm-1 indicates the presence of aliphatic C-H bonds, confirming alkane
chains.
 1607 cm-1 and 1447 cm-1 strongly suggest aromatic C=C stretching, indicative
of an aromatic ring.
 1044 cm-1 corresponds to C-O stretches, suggesting the presence of oxygen-
containing functional groups (e.g., alcohols).
 The lower wavenumbers (873 and 822 cm -1) further confirm aromatic out-of-
plane bending vibrations.

Fig. 4-Hydroxyisophalic acid Fig. 2-hydroxy 4-methoxy benzaldehyde

IR Interpretation of Plumbago zeylanica.

WAVE
NUMBER SIGNAL INTERPRETATION
(cm-1)

3425.05 O-H stretching (broad, likely from alcohol ).

2924.82,
C-H stretching vibrations (aliphatic C-H, sp³).
2855.15

2058.29 Possible combination band or overtone.

1625.45,
C=C stretching (aromatic ).
1517.66

1448.21 C-H bending (methyl or methylene groups).

1384.07 C-H deformation

1238.75,
C-O stretching .
1057.14

823.44 C-H out-of-plane bending (aromatic substitution).

631.72 Likely skeletal vibrations

Table No. 4 – Interpretation of IR Spectrum of Plumbago zeylanica.

Interference :

1238.75 cm-1: This peak corresponds to C-O stretching vibrations, which are
commonly seen in esters, ethers, or alcohols. The overall interpretation remains
consistent, the spectrum indicates a compound containing:

 O-H groups (alcohols, phenols),

 Aliphatic C-H bonds,
 Aromatic or unsaturated (C=C) structures,
 C-O bonds (suggesting esters, ethers, or alcohols).

Conclusion :

 O-H Stretch (3425 cm-1): Indicates the presence of hydroxyl groups, possibly
alcohols, phenols, or water.
 C-H Stretch (2924, 2851 cm- ): Suggests aliphatic C- H bonds.
 C = C Stretch (1625cm -1) Implies the presence of aromatic or unsaturated
(alkene) groups.
 C-O Stretch (1236, 1057 cm -1 ) : Strong signals pointing to C-O bonds, often
found in esters, ethers, or alcohols.
 Aromatic Signals (823 cm-1): Out-of-plane bending vibrations suggest aromatic
substitution.

Fig. Structure of Plumbagin

FORMULATION PROCEDURE

[Link] INGREDIENTS FUNCTION QUANTITY

Liquid Paraffin Emollient 7.5ml

1

2 Bees Wax Emulsifier/Thickener 4gm

3 Glycerin Humectant 1ml

4 Water Aqueous phase 9ml

5 Borax Stabilising agent 0.1gm

Decalepis hamiltonni root

6 Active ingredient 2ml
extract

Plumbago zeylanica root

7 Active ingredient 2ml
extract

8 Sodium benzoate Preservative 0.125gm

9 Sandalwood oil Fragrance 3dps

Table No.5 - Ingredients required for Cream Formulation.

1. Preparation of Oil Phase:

In a clean, china Dish add (Liquid paraffin or mineral oil), beeswax. Heat the mixture
to 70–75°C until the wax is completely melted. Stir the mixture continuously to ensure
uniform blending.

2. Preparation of Water Phase:

In another beaker, heat distilled water to 70–75°C. Add glycerin to the water and mix
until it dissolves completely.

3. Combining Oil and Water Phases:

Slowly add the heated water phase to the oil phase while stirring continuously. Use a
homogenizer or a mechanical stirrer to create a uniform emulsion.

4. Cooling and Addition of Active Ingredients:

Allow the emulsion to cool to about 40°C with continuous stirring. Add the ethanolic
root extracts of Plumbago zeylanica and Decalepis hamiltonii to the emulsion. Mix
thoroughly to ensure even distribution of the active ingredients.

5. Adjust pH:

Measure the pH of the formulation (ideal range: 5–6). If necessary, adjust using a
small amount of citric acid (to lower pH) or sodium hydroxide solution (to raise pH).

6. Addition of Preservative and Fragrance:

Add the preservative and mix well. If desired, incorporate a small amount of fragrance
for sensory appeal.

7. Final Mixing and Packaging:

Continue stirring until the cream reaches room temperature and has a uniform, smooth
texture. Transfer the cream into sterilized, airtight containers. Label the containers
with product information and storage instructions.
Fig. No. 6 Cream Formulation from the extracts of Plumbago zeylanica and
Decalepis hamiltonii
EVALUATION OF ANTIMICROBIAL CREAM

The evaluation of an antimicrobial cream was performes to assess its physicochemical

properties, stability, and biological activity. Below are the key parameters:
1. Organoleptic Properties

 Appearance: The cream had a uniform color and texture.

 Odor: It had a pleasant or neutral smell without any off-odors.
 Consistency: The cream had a good spreadability on the skin without being
too greasy or too runny.
2. pH Measurement

The pH of the cream formulation was measured using pH paper and found to be 6.2
This indicates that the cream is slightly acidic, which is suitable for topical application
as it aligns closely with the natural pH of the skin (typically between 4.5 and 6.5).
Maintaining a pH close to the skin’s natural level helps in minimizing irritation and
maintaining the skin barrier function.

3. Stability Testing

Stability tests was conducted under various conditions (temperature, light, humidity)
to ensure that the cream retains its properties over time.

[Link]

The formulation was found to have assess ease of removal with water.
PRINCIPLE

Known concentration of Ciprofloxacin and Nystatin antibiotic discs were on

agar plate that has been inoculated or seeded uniformly over the entire plate with a
culture of the bacterium to be tested. The plate extract inoculated for 18-24 hours at
37°C. During this period the antibacterial agents diffuses through the agar and may
prevent the growth of organism. Effectiveness of susceptibility is proportional to the
diameter of zone of inhibition around the disc organism which grows up to the edge of
the disc are resistant.
MATERIALS
 Incubator
 Sterilised discs
 Zone reader
 Nutrient broth
MEDIAS
 Media for bacteria - Nutrient agar
 Media for fungi - Sabouraud dextrose agar
ORGANISM USED
Bacteria - Gram positive
 Staphylococcus aureus
Bacteria - Gram negative

 Escherichia coli
Fungi
 Aspergillus niger
 Candidias albicans

ANTIBIOTIC DISCS
Ciprofloxacin 5μg/disc

Nystatin 100units/disc
 TEST SAMPLE

Extracts of roots and their combination.

Medium preparation procedure

 Sabouraud Dextrose Agar (Plating medium)

This is the most useful selective medium for the culture of mycotic agents ,
particularly the filamentous moulds. With the addition of antibiotics (chloramphenicol
or cycloheximide, or a combination of pencillin and streptomycin), growth of bacterial
contaminants can be prevented.

Ingredients
Dextrose 40 g
Peptone 10 g
Agar 15 g
Distilled water 1000 ml

 Dissolve agar in 1000 ml of distilled water by heating.

 While hot , add peptone and dextrose
 Boil gently until dissolved
 Adjust the PH 6.0
 Dispense into culture tubes (20-ml) with cotton plugs or caps
 Sterilize by autoclaving (1210 c for 15 minutes)
 Cool the culture medium in slants , or when the temperature of the medium
reaches 500 c, pour in sterilized plates
 Sabouraud Dextrose Broth (sub culturing medium)

Ingredients
Dextrose 40 g
Peptone 10 g
Distilled water 1000 ml
 Dissolve the ingredients with gentle heating and stirring.
 Dispense in 10 ml amounts in culture tubes.
 Autoclave ( 121 0 c for 10 minutes).

ANTI-FUNGAL ACTIVITY PROCEDURE

The ATCC numbered strains bought from Madaras Medical College was
periodically sub cultured in Sabouraud dextrose agar and maintained in the
laboratory. The Strains namely Aspergillus niger & Candida albicans were
brought to the active phase by sub culturing in Sabouraud dextrose broth and
incubated at room temperature for 4 days. The Standardized inoculam about 0.1
ml was inoculated on Sabouraud dextrose agar uniformly. The sterile disc (watt
man No.2 of 6 mm diameter) was placed at equal interval on uniformly inoculated
plate and a standard disc Nystatin 100 units/disc was also placed by aseptic
technique. The test sample about 100 µl was loaded to the sterile disc by using
aseptic precautions. The plates were incubated at room temperature for 2 to 4
days. During this period the drug diffuse through the agar and inhibit the growth if
the drug is potent. The diameter of inhibiting zone around the disc was measured
by using the ruler.

Interpretation has been assessed based on the following table

[Link] Zone of Inhibition in mm Report

1. Zone of inhibition between 6mm to 12mm Intermediate

2. Zone of inhibition below 6mm Resistance

3. Zone of inhibition more than 12mm Sensitive

Report on Antifungal Activity of the the given samples

The anti-fungal activity for the given sample was carried out by Disc Diffusion
Technique (Indian Pharmacopoeia 1996, Vol II A-105). The test microorganisms of
Aspergillus niger and Candida albicans, were maintained by periodical sub culturing
on Sabouraud dextrose agar medium . The effect produced by the sample was
compared with the effect produced by the positive control (Reference standard
Nystatin 100 µg/disc for fungi).

The obtained results are tabulated as follows

Zone of inhibition in mm
[Link] Name of the organism
PZ DH BO STANDARD

1. [Link] 15 15 24 30

2. [Link] 12 18 22 26

Table No. 6 Report on Antifungal Activity of the the given samples

PZ - Plmbago zeylanica (root extract)

DH - Decalepis hamiltonni (root extract)

BO - Combined root extract of Plumbago zeylanica and Decalepis hamiltonni

STANDARD - Nystatin 100 units/disc for fungi.

ANTI BACTERIAL ACTIVITY PROCEDURE

The NCIM numbered strains bought from National Chemical Laboratory

(NCL) Pune was periodically sub cultured in Nutrient agar and maintained in the
laboratory. The Strains namely Staphylococcus aureus and [Link] were brought to the
active phase by sub culturing in Nutrient broth and incubated at 37 0c for 18 hours.
The Standardized inoculam about 0.1 ml was inoculated on Muller hinton
agar(Hi media) uniformly. The sterile disc (watt man No. 2 of 6 mm diameter was
placed at equal interval on uniformly inoculated plate and a standard disc
Ciprofloxacin 5 mcg/disc was also placed by aseptic technique. The test sample about
100 µl was loaded to the sterile disc by using aseptic precautions. The plates were
incubated at 37 0 C for 24 hours. During this period the drug diffuse through the agar
and inhibit the growth if the drug is potent. The diameter of inhibiting zone around the
disc was measured by using the ruler and

Interpretation has been assessed based on the following table.

[Link] Zone of Inhibition Report

1. Zone of inhibition between 6mm to 12mm Intermediate

2. Zone of inhibition below 6mm Resistent

3. Zone of inhibition more 12mm Sensitive

Report on Antibacterial Activity of the the given samples

The anti-microbial activity for the given sample was carried out by Disc
Diffusion Technique (Indian Pharmacopoeia 1996, Vol II A-105). The test
microorganisms of Staphylococcus aureus and [Link] were obtained from National
Chemical Laboratory (NCL) Pune and maintained by periodical sub culturing on
Nutrient agar and Sabouraud dextrose agar medium for bacteria and Fungi
respectively. The effect produced by the sample was compared with the effect
produced by the positive control (Reference standard Ciprofloxacin 5 µg/disc for
bacteria).

The obtained results are tabulated as follows:

Zone of inhibition in mm
Name of the
[Link]
microorganisms
PZ DH BO STANDARD

1. [Link] 18 20 25 33

2. Staphylococcus aureus 29 20 31 20

Table No. 7 Report on Antibacterial Activity of the the given samples

PZ - Plmbago zeylanica (root extract)

DH - Decalepis hamiltonii (root extract)

BO - Combined root extract of Plumbago zeylanica and Decalepis hamiltonii

STANDARD - Ciprofloxacin 5μg/disc for bacteria.

[Link].7 Resistance against Candidias albicans

[Link].8 Resistance against Aspergillus niger

 [Link].9 Resistance against [Link]

[Link].10 Resistance against Staphylococcus aureus

RESULT AND DISCUSSION

Preliminary phytochemical investigation of alcoholic extracts shows the presence of

Alkaloids , Carbohydrates, Flavonoids, Cardiac Glycosides, Terpenoids, Steroids and
Tannins . In the present study the spectral analysis of extracts were subjected to UV
and the maximum absorption for Decalepis hamiltonii (DH) was observed at 205.1
nm, 220.5nm, 279.9nm, 680.7 nm and for Plumbago zeylanica (PZ) at 210nm,
220.6nm 274.9nm . Alcoholic extract were subjected to FTIR studies and functional
group responsible for absorption are listed in the table 3 & 4.

The antimicrobial activity of Plumbago zeylanica (PZ), Decalepis hamiltonii (DH),

and their combination (BO) was evaluated against bacterial and fungal strains.
Ciprofloxacin and Nystatin were used as standards for comparison.

Antibacterial Activity against Escherichia coli

 PZ showed a Zone of Inhibition (ZOI) of 18 mm

 DH exhibited slightly higher activity with a ZOI of 20 mm
 BO demonstrated significantly enhanced activity with a ZOI of 25 mm,
suggesting a synergistic effect.
 Ciprofloxacin (standard) had a ZOI of 33 mm, indicating that the combination
(BO) displayed moderate efficacy compared to the standard
Antibacterial Activity against Staphylococcus aureus

 PZ showed a ZOI of 29 mm
 DH exhibited a ZOI of 20 mm
 BO demonstrated the highest activity with a ZOI of 31 mm, surpassing the
standard ciprofloxacin 20 mm
Antifungal Activity against Candida albicans

 Both PZ and DH individually exhibited moderate activity with ZOI values of

15 mm each
  BO showed a marked improvement in activity with a ZOI of 24 mm, indicating
synergism
 Nystatin (standard) showed a ZOI of 30 mm, which is higher than BO but still
indicates good efficacy for the combination
Antifungal Activity against Aspergillus niger:

 PZ showed minimal activity with a ZOI of 12 mm

 DH exhibited higher activity with a ZOI of 18 mm
 BO demonstrated significant enhancement with a ZOI of 22 mm, indicating
synergistic action.
 Nystatin (standard) had a ZOI of 26 mm, showing that BO possesses
considerable antifungal potential.

The combination of PZ and DH (BO) demonstrated enhanced antimicrobial activity

compared to the individual extracts for all tested microorganisms. This suggests a
synergistic interaction between the phytoconstituents of the two extracts, resulting in
improved efficacy. The enhanced activity could be attributed to because of the
improved penetration and disruption of microbial cells due to the synergistic effect.
This highlights its potential as a natural antimicrobial agent, particularly in the
formulation of creams or other topical applications. Further studies, including in vivo
evaluations and testing of the cream formulation, are recommended to confirm its
therapeutic potential in future.
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RYUH