Tulsi: An important medicinal plant

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Tulsi: An important medicinal plant

1.1 General introduction:

History of many drugs can be traced back to their natural origin. At the very dawn of civilization, coexistence of disease with the emergence of life in the earth compelled the primitive man to search a cure from his surroundings. Since then plant was being used as remedy for many diverse disease ranging from simple skin infection to such formidable foes as heart diseases and cancers. The medicinal use of some plants by Indo-Aryans was noted in the Rig Veda (4500-1600BC) and the medicinal value of many plant constituents used by Egyptians was recorded in Papyrus Abus (1500BC).The great physician Hippocrates, who is called ‘the father of medicinal science’, also used plants for treatment of many diseases. In this respect medicine can be considered as an ancient art consisting of plant materials.¹

Starting from the stone-age, the use of plants as traditional medicine has increased with the age of civilization. Herbal medicines are still in use in many societies. Although most of the modern medicines are simple compounds, but in many cases the drugs have been originated from the nature, more specifically from plant sources. Plants are considered as natural chemical factory, because synthetic process in biological systems particularly in plants is going on by nature’s ordinary condition of temperature and pressure. The laboratory synthesis of anti-malarial drug quinine requires an intensive work extending over half a century but chicona plant can do it everyday without difficulty. Till today, extensive phytochemical analysis of plants yielded diversified chemical compounds as steroids, terpenoids, flavonoids, chalcones, alkaloids, glycosides etc. clinically used plant metabolites such as taxol isolated from Taxes brevifolia, Vineristine and Vinblastine obtained from Vinca rosea Linn. and digitalis derived from Digitalis purpura are only few of the many striking examples of developing life saving drugs from plant sources.²

Once the plant medication was provided to the ancient people in the crude form, it often exhibited many unwanted effects due to the presence of some toxic components beyond the active constituent. Extensive phytochemical investigation and isolation of active component in the pure form thus became necessary to avoid untoward effects and to ensure safe use of herbal medicines, phytochemical studies of medicinal plants got a rapid pace and the presence of many chemical compounds of diversified nature including many new compounds came in light. These plant derived compounds often played an important role in directing laboratory synthesis of many new classes of drug molecules. In some cases, the plant components became the starting material in the synthetic process of industrial production of many drug molecules. For example, the use of sterol diosgenin, isolated from Mexican yam, for laboratory synthesis of oral contraceptive progesterone reduce the cost of progesterone from a value of $80 per gram to $1.75 per gram.³

Sometimes phytochemical analysis for many plants yielded such chemical constituent having no remarkable therapeutic interest. Sometimes the crude drugs containing several constituents were found to be ineffective in case of therapy for which it is used. The phytochemical investigation of periwinkle plant (Vinca rosea), once used traditionally as an anti-diabetic drug, was found to contain alkaloidal constituents having hypoglycemic potency in minute quantities. The dried seeds of plant Amni visanaga was used as diuretic and antispasmodic in renal collie in Eastern Mediterranean countries and in Arabia, but the research carried out by G.V. Anrep and coworkers resulted in the isolation of Khelin, a component having vasodilator effect. Khelin appeared in an anti-anginel drug after subsequent clinical trial. The research on Ranwolfia serpentine, which was traditionally used as an antidote for snake bite, revealed the presence of an antihypertensive agent reserpine. Thus systemic research with medicinal plant might open the door of many unknown therapeutic choices. Serendipity has also much impact in discovery of drug leads from natural origin.⁴

From the above discussion it is evident that the search of plant constituents having therapeutic interest requires bioactivity studies with the crude extracts prior to phytochemical investigation. Only the bioactive extracts or fraction would be of interest for next phytochemical analysis. Without prior biological studies, the phytochemical analysis alone can provide the chemical composition of the plant which may or may not have therapeutic interest. Thus in conducting research on medicinal plants, bioactivity guided phytochemical analysis might be a rational approach.⁵

1.2 Plant Based Drugs and Medicines:

Today there are at least 120 distinct chemical substances derived from plants that are considered as important active compounds for preparation of drugs currently in use in one or more countries in the world. These chemical substances are shown in the table below. Several of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The original plant substance/chemical name is shown under the “Drug” column rather than the finished patented drug name. For example, many years ago a plant chemical was discovered in a tropical plant, Cephaelis ipecacuanha, and the chemical was named emetine.⁶ a drug was developed from this plant chemical called Ipecac which was used for many years to induce vomiting mostly if someone accidentally swallowed a poisonous or harmful substance. Ipecac can still be found in pharmacies in many third world countries but has been mostly replaced by other drugs in the United States. Another example of this is the plant chemical named taxol shown in the drug column below. The name taxol is the name of the plant chemical originally discovered in the plant. A pharmaceutical company copied this chemical and patented a drug named Paclitaxe which is used in various types of tumors today in the U.S. and many other countries.^7-8

The 120 substances given in table-1 are sold as drugs worldwide but not in all countries. Some European countries regulate herbal substances and products differently than in the United States. Many European countries, including Germany, regulate herbal products as drugs and pharmaceutical companies prepare plant based drugs simply by extracting out the active chemicals from the plants.⁹

Some of the drug/chemicals shown in table-1 are still sold as plant based drugs requiring the processing of the actual plant material. Others have been chemically copied or synthesized by laboratories and no plant materials are used in the manufacture of the drug. A good example of this is the plant chemical quinine, which was discovered in a rainforest tree (Cinchona ledgeriana) over 100 years ago. For many years the quinine chemical was extracted from the bark of this tree and processed into pills to treat malaria. Then a scientist was able to synthesize or copy this plant alkaloid into a chemical drug without using the original tree bark for manufacturing the drug. Today, all quinine drugs sold are manufactured chemically without the use of any tree bark. However, another chemical in the tree called quinidine which was found to be useful for various heart conditions couldn’t be completely copied in the laboratory and the tree bark is still harvested and used to extract this plant chemical from it. Quinidine extracted from the bark is still used today to produce quinidine-based drugs.¹⁰

Table-1: List of drugs or chemicals of plant origin having clinical use.

Drug/Chemical	Action/Clinical Use	Plant Source
Acetyldigoxin	Cardiotonic	Digitalis lanata
Adoniside	Cardiotonic	Adonis vernalis
Aescin	Anti-inflammatory	Aesculus hippocastanum
Aesculetin	Anti-dysentery	Frazinus rhychophylla
Agrimophol	Anthelmintic	Agrimonia supatoria
Ajmalicine	Circulatory Disorders	Rauvolfia sepentina
Allantoin	Vulnerary	Several plants
Allyl isothiocyanate	Rubefacient	Brassica nigra
Anabesine	Skeletal muscle relaxant	Anabasis sphylla
Andrographolide	Baccillary dysentery	Andrographis paniculata
Anisodamine	Anticholinergic	Anisodus tanguticus
Anisodine	Anticholinergic	Anisodus tanguticus
Arecoline	Anthelmintic	Areca catechu
Asiaticoside	Vulnerary	Centella asiatica
Atropine	Anticholinergic	Atropa belladonna
Benzyl benzoate	Scabicide	Several plants
Berberine	Bacillary dysentery	Berberis vulgaris
Bergenin	Antitussive	Ardisia japonica
Betulinic acid	Anticancerous	Betula alba
Borneol	Antipyretic, analgesic, anti-inflammatory	Several plants
Bromelain	Anti-inflammatory, proteolytic	Ananas comosus
Caffeine	CNS stimulant	Camellia sinensis
Camphor	Rubefacient	Cinnamomum camphora
Camptothecin	Anticancerous	Camptotheca acuminata
(+)-Catechin	Haemostatic	Potentilla fragarioides
Drug/Chemical	Action/Clinical Use	Plant Source
Chymopapain	Proteolytic, mucolytic	Carica papaya
Cissampeline	Skeletal muscle relaxant	Cissampelos pareira
Cocaine	Local anaesthetic	Erythroxylum coca
Codeine	Analgesic, antitussive	Papaver somniferum
Colchiceine amide	Antitumor agent	Colchicum autumnale
Colchicine	Antitumor agent, anti-gout	Colchicum autumnale
Convallatoxin	Cardiotonic	Convallaria majalis
Curcumin	Choleretic	Curcuma longa
Cynarin	Choleretic	Cynara scolymus
Danthron	Laxative	Cassia species
Demecolcine	Antitumor agent	Colchicum autumnale
Deserpidine	Antihypertensive, tranquillizer	Rauvolfia canescens
Deslanoside	Cardiotonic	Digitalis lanata
L-Dopa	Anti-parkinsonism	Mucuna sp
Digitalin	Cardiotonic	Digitalis purpurea
Digitoxin	Cardiotonic	Digitalis purpurea
Digoxin	Cardiotonic	Digitalis purpurea
Emetine	Amoebicide, emetic	Cephaelis ipecacuanha
Ephedrine	Sympathomimetic, antihistamine	Ephedra sinica
Etoposide	Antitumor agent	Podophyllum peltatum
Galanthamine	Cholinesterase inhibitor	Lycoris squamigera
Gitalin	Cardiotonic	Digitalis purpurea
Glaucarubin	Amoebicide	Simarouba glauca
Glaucine	Antitussive	Glaucium flavum
Glasiovine	Antidepressant	Octea glaziovii
Glycyrrhizin	Sweetener, Addison’s disease	Glycyrrhiza glabra
Gossypol	Male contraceptive	Gossypium species
Drug/Chemical	Action/Clinical Use	Plant Source
Hemsleyadin	Bacillary dysentery	Hemsleya amabilis
Hesperidin	Capillary fragility	Citrus species
Hydrastine	Hemostatic, astringent	Hydrastis Canadensis
Hyoscyamine	Anticholinergic	Hyoscyamus niger
Irinotecan	Anticancer, antitumor agent	Camptotheca acuminata
Kaibic acud	Ascaricide	Digenea simplex
Kawain	Tranquillizer	Piper methysticum
Kheltin	Bronchodilator	Ammi visage
Lanatosides A, B, C	Cardiotonic	Digitalis lanata
Lapachol	Anticancer, antitumor	Tabebuia sp.
a-Lobeline	Smoking deterrant, respiratory stimulant	Lobelia inflate
Menthol	Rubefacient	Mentha species
Methyl salicylate	Rubefacient	Gaultheria procumbens
Monocrotaline	Antitumor agent (topical)	Crotalaria sessiliflora
Morphine	Analgesic	Papaver somniferum
Neoandrographolide	Dysentery	Andrographis paniculata
Nicotine	Insecticide	Nicotiana tabacum
Nordihydroguaiaretic acid	Antioxidant	Larrea divaricata
Noscapine	Antitussive	Papaver somniferum
Ouabain	Cardiotonic	Strophanthus gratus
Pachycarpine	Oxytocic	Sophora pschycarpa
Palmatine	Antipyretic, detoxicant	Coptis japonica
Papain	Proteolytic, mucolytic	Carica papaya
Papavarine	Smooth muscle relaxant	Papaver somniferum
Phyllodulcin	Sweetner	Hydrangea macrophylla
Physostigmine	Cholinesterase Inhibitor	Physostigma venenosum
Picrotoxin	Analeptic	Anamirta cocculus
Drug/Chemical	Action/Clinical Use	Plant Source
Pilocarpine	Parasympathomimetic	Pilocarpus jaborandi
Pinitol	Expectorant	Several plants
Podophyllotoxin	Antitumor anticancer agent	Podophyllum peltatum
Protoveratrines A, B	Antihypertensives	Veratrum album
Pseudoephredrine*	Sympathomimetic	Ephedra sinica
Pseudoephedrine, nor-	Sympathomimetic	Ephedra sinica
Quinidine	Antiarrhythmic	Cinchona ledgeriana
Quinine	Antimalarial, antipyretic	Cinchona ledgeriana
Qulsqualic acid	Anthelmintic	Quisqualis indica
Rescinnamine	Antihypertensive, tranquillizer	Rauvolfia serpentine
Reserpine	Antihypertensive, tranquillizer	Rauvolfia serpentine
Rhomitoxin	Antihypertensive, tranquillizer	Rhododendron molle
Rorifone	Antitussive	Rorippa indica
Rotenone	Piscicide, Insecticide	Lonchocarpus nicou
Rotundine	Analagesic, sedative, traquillizer	Stephania sinica
Rutin	Capillary fragility	Citrus species
Salicin	Analgesic	Salix alba
Sanguinarine	Dental plaque inhibitor	Sanguinaria Canadensis
Santonin	Ascaricide	Artemisia maritma
Scillarin A	Cardiotonic	Urginea maritime
Scopolamine	Sedative	Datura species
Sennosides A, B	Laxative	Cassia species
Silymarin	Antihepatotoxic	Silybum marianum
Sparteine	Oxytocic	Cytisus scoparius
Drug/Chemical	Action/Clinical Use	Plant Source
Stevioside	Sweetner	Stevia rebaudiana
Strychnine	CNS stimulant	Strychnos nux-vomica
Taxol	Antitumor agent	Taxus brevifolia
Teniposide	Antitumor agent	Podophyllum peltatum
a-Tetrahydrocannabinol

(THC)Antiemetic, decrease occular tensionCannabis sativaTetrahydropalmatineAnalgesic, sedative, traquillizerCorydalis ambiguaTetrandrineAntihypertensiveStephania tetrandraTheobromineDiuretic, vasodilatorTheobroma cacaoTheophyllineDiuretic, bronchodilatorTheobroma cacao and othersThymolAntifungal (topical)Thymus vulgarisTopotecanAntitumor, anticancer agentCamptotheca acuminataTrichosanthinAbortifacientTrichosanthes kirilowiiTubocurarineSkeletal muscle relaxantChondodendron tomentosumValapotriatesSedativeValeriana officinalisVasicineCerebral stimulantVinca minorVinblastineAntitumor, Antileukemic agentCatharanthus roseusVincristineAntitumor, Antileukemic agentCatharanthus roseusYohimbineAphrodisiacPausinystalia yohimbeYuanhuacineAbortifacientDaphne genkwaYuanhuadineAbortifacientDaphne genkwa

1.3 Role of Plant on Cancer and Aids Research:

The National Cancer Institute (NCI) has several ongoing collaborative programs which screen plants for the possibility of new drugs and active plant chemicals for cancer and AIDS/HIV.

As well over 50 percent of the estimated 250,000 plant species found on earth come from tropical forests, NCI concentrates on these regions. Plants have been collected from the African countries of Cameroon, the Central African Republic, Gabon, Ghana, Madagascar, and Tanzania. Collections are now concentrated in Madagascar (one of the most rapidly disappearing rainforest regions in the world), and collaborative programs have been established in South Africa and Zimbabwe.

In Central and South America, samples have been collected from Belize, Bolivia, Colombia, the Dominican Republic, Ecuador, Guatemala, Guyana, Honduras, Martinique, Paraguay, Peru, and Puerto Rico. The NCI has established collaborative programs in Brazil, Costa Rica, Mexico, and Panama. Southeast Asian collections have been performed in Bangladesh, Indonesia, Laos, Malaysia, Nepal, Pakistan, Papua New Guinea, the Philippines, Taiwan, Thailand, and Vietnam. Collaborative programs have been established in Bangladesh, China, Korea, and Pakistan. In each country, NCI contractors work in close collaboration with local botanical institutions.¹⁴

Thus far seven plant-derived anticancer drugs have received Food and Drug Administration (FDA) approval for commercial production: ¹⁵

• Taxol/paclitaxel

A chemical discovered in the Pacific Yew tree (Taxus brevifolia) is now the first drug of choice in several tumorous cancers including Breast Cancer.

• Vinblastine

A chemical discovered in the Madagascar Periwinkle in the 1950s. Vinblastine is the first drug of choice in many forms of leukemia and since the 1950’s it has increased the survival rate of childhood leukemias by 80%.

• Vincristine

Another antileukemic drug discovered in the Madagascar periwinkle.

• Topotecan

It has been approved by the FDA for the treatment of ovarian and small cell lung cancers. It is currently in clinical trials, either alone or in combination with other anticancer drugs, for several types of cancer. Topotecan is an analog (a synthesized chemical) of a plant alkaloid discovered in the Chinese tree species, Camptotheca acuminata.

• Irinotecan

Another chemical analog which has been developed from yet another plant alkaloid discovered in the same tree Camptotheca acuminata. It has been approved by the FDA for the treatment of metastatic colorectal cancer. It is currently in clinical trials for a variety of other cancers.

• Etoposide

It is a semi synthetic derivative of a plant chemical epipodophyllotoxin discovered in the May apple plant family (Podophyllum peltatum).

• Teniposide

It is another semi synthetic derivative of a plant chemical discovered in the May apple plant family (Podophyllum peltatum).

Since 1986, over 40,000 plant samples have been screened, but thus far only five chemicals showing significant activity against AIDS have been isolated. Three are currently in preclinical development. Before being considered for clinical trials in humans, these agents must show tolerable levels of toxicity in several animal models. For AIDS, three agents are presently in preclinical or early clinical development. The following are plants and chemicals which are still under research for cancer and AIDS/HIV:

• (+)-Calanolide A and (-)-Calanolide B (costatolide) are isolated from Calophyllum lanigerum and Calophyllum teysmanii, respectively, trees found in Sarawak, Malaysia. Both these agents are licensed to Medichem, Inc., Chicago, which is developing them in collaboration with the Sarawak State Government through a joint company, Sarawak Medichem Pharmaceuticals, Inc.(+)-Calanolide A is currently in early clinical trials in the United States.

• Conocurovone, isolated from the shrub species, Conospermum incurvum (saltbush), found in Western Australia, has been licensed for development to AMRAD, a company based in Victoria, Australia.

• Michellamine B, from the leaves of Ancistrocladus korupensis, a vine found in the Korup rainforest region of southwest Cameroon, has undergone extensive preclinical study, but is considered too toxic for advancement to clinical trials.

• Prostratin, isolated from the wood of Homolanthus nutans, a tree found in Western Samoa, has been placed on low priority, largely due to its association with a class of compounds shown to be tumor promoters.

• A tree native to China, Camptotheca acuminate, is the source of four promising anticancer drugs, two of which have been approved by the FDA and are described above. The other two chemicals still under research include:

• 9AC (9-aminocamptothecin): Currently in clinical trials for several types of cancer, including ovarian and stomach cancers and T-cell lymphoma.

• Camptothecin: While no clinical trials are being performed in the United States, trials are ongoing in China.

• Homoharringtonine from the Chinese tree, Cephalotaxus harringtonia are in early clinical trials.

• Perillyl alcohol, and flavopiridol, a totally synthetic compound based on a flavone isolated from Dysoxylum binectiferum are in early clinical trials.^16-17

1.4 Bioactivity guided phytochemical investigation of the plants:

The use of plant products is increasing in many segment of the population. At present thousands of plant metabolics are being successively used in the treatment of variety of diseases. According to estimation 40% of the world’s populations rely upon plants for their medication. The use of medicinal plants is increasing in many developed countries where 35% of drugs involve natural products. Since Bangladesh has a vast resource of medicinal plants, the present study might be a significant way of making the best use of these natural resources. Majority of our population, who are impoverished, have to rely upon indigenous system of medication because of their inability to meet the cost of modern medicine. Thus in order to strengthen the existing health care system, biological activity directed chemical analysis of indigenous plant Ocimum sanctum is the primary objective of bioactivity directed phytochemical investigation of plant products. Moreover the standardization of herbal medicines has made them popular in many developed countries. So the economic impact of the present study may be reflected through export standard, high quality herbal drugs, which would increase our natural reserve. Thus the rationality of the present study lies in meeting the challenge of developing herbal medicine for the coming twenty first century which needs a systematic research on indigenous medicinal plants for the welfare of the humanity.

1.5 Objective:

Main objectives of the proposed research program are:

1. To extract and isolate biologically active compounds from the leaves of O. sanctum using various solvents such as methanol, chloroform, ethyl acetate, n-hexane and butanol and to identify the structure of the isolated compounds using various analytical and instrumental techniques including UV, IR, ¹H-NMR, ¹³C-NMR and mass spectroscopy.

2. To evaluate the medicinal value of the Isolated Compounds obtained from the extracts of the leaves of O. sanctum by means of several studies related to antibacterial, antifungal.

3. To identify and suggest the possible area of the utilization of the isolated compounds from the extract of seeds in the cure of various diseases.

1.6 Literature Survey:

1.7 General Information:

Kingdom	: Plantae
Subkingdom	: Tracheobionta
Superdivision	: Spermatophyta
Division	: Magnoliophyta
Class	: Magnoliopsida
Order	: Lamiales
Family	: Lamiaceae
Genus	: Ocimum
Species	: O. sanctum

Indian Name:	Tulsi
Botanical or Latin Name :	Ocimum sanctum
English name :	Sacred Basil / Holy Basil

1.8 Origin of Ocimum sanctum:

Holy basil is native to tropical Asia but has been dispersed by humans so that it now grows in many tropical parts of the world. It is a sacred plant in Hindu religion, and has been cultivated in India in courtyards or temples, and in pots in homes, for about 3000 years.

Holy basil has been cultivated in India for thousands of years.

1.9 History:

The history of the plant in South Asia is closely linked with folklore and mythology. It represents Vishnupriya or Beloved of Vishnu, since it is believed to be the embodiment of the goddess Lakshmi, the spouse of Vishnu. What is apparent is that it has been valued and cultivated since ancient times in India as an intimate link between the household and the spiritual world.

The Aryans, who structured the forms of Hinduism, were nature-worshippers and their poetry and imagery were rich with the evocation of nature. Perhaps they were drawn to holy basil because of its fragrance and delicacy. It may also have been already well-entrenched in the myths of the indigenous people and from there absorbed into Hinduism.

Holy basil is mentioned in the Rig Veda, written in about 1500 BC, and its holiness is celebrated in the Puranas. It is highly regarded in the Ayurvedic system of medicine and is noted in medical treatises such the Charaka Samhita written between the 2nd century BC to the 2nd century AD.

1.10 Description of OCIMUM SANCTUM:

Tulsi plant, is a shrub reaching a height of 0.5 to 1.5 m. The leaves are 2-4 cms in length. There are several varieties of the plant. However, commonly used one is with dark leaves. The inflorescence is a long spike with tiny purple flowers. There are two varieties: a red- and a green one. Red holy basil has a stronger smell. It is cultivated in gardens throughout Bengal, /East Nepal and Deccan Peninsula; it is said to be a common wild plant in Western India.

1.11 Characteristics of Constituents:

The leaves contain an essential oil which has been studied with gas chromatography. The oil contains eugenol, eugenal, carvacrol, methylchavicol, limatrol and caryophylline. The seeds contain an oil composed of fatty acids and sitosterol. The mucilage is compared of sugars – xylose and polysaccharides.

Tulasi as an Ayurvedic medicine

Tulasi, as used in Ayurveda.

Tulasi’s extracts are used in ayurvedic remedies for common colds, headaches, stomach disorders, inflammation, heart disease, various forms of poisoning, and malaria. Traditionally, tulasi is taken in many forms: as an herbal tea, dried powder, fresh leaf, or mixed with ghee. Essential oil extracted from Karpoora Tulsi is mostly used for medicinal purposes and in herbal toiletry. For centuries, the dried leaves of Tulasi have been mixed with stored grains to repel insects.

Recent studies suggest that Tulasi may be a COX-2 inhibitor, like many modern painkillers, due to its significant amount of Eugenol (1-hydroxy-2-methoxy-4-allylbenzene).^{<href=”#_note-4″ title=””>[5]<href=”#_note-5″ title=””>[6]} Studies have also shown Tulsi to be effective for diabetes, by reducing blood glucose levels.^{<href=”#_note-6″ title=””>[7]} The same study showed significant reduction in total cholesterol levels with Tulsi. Another study showed that Tulsi’s beneficial effect on blood glucose levels is due to its antioxidant properties.^{<href=”#_note-7″ title=””>[8]}

Tulasi also shows some promise for protection from radiation poisoning^{<href=”#_note-8″ title=””>[9]} and cataracts.^{<href=”#_note-9″ title=””>[10]} Some Vaishnavites do not use Tulasi for medicine, though, out of reverence. However, the use of Tulsi for purification and as a medicine is widespread throughout India. Many Hindus — along with the ancient tradition of Ayurveda — believe that the healing properties of sacred herbs such as Tulsi were given by the Lord himself, and can be used as a medicine out of reverence.

1.12 Parts used and where grown

Holy basil is native to the Indian subcontinent and other parts of tropical Asia. The leaf and seed oil are used therapeutically.

Holy basil has been used in connection with the following conditions (refer to the individual health concern for complete information):

Science Ratings	Health Concerns
	Asthma

Type 2 diabetes Poison oak and poison ivy dermatitisReliable and relatively consistent scientific data showing a substantial health benefit.

Contradictory, insufficient, or preliminary studies suggesting a health benefit or minimal health benefit.

For a herb, supported by traditional use but minimal or no scientific evidence. For a supplement, little scientific support and/or minimal health benefit.

1.13 Varieties of Holy Basil-Tulsi:

Krishna Tulsi Rama Tulsi Vana Tulsi

1.14 Active compounds:

The essential oil from some populations of holy basil contains high levels of eugenol. This compound has anti-inflammatory activity, can kill bacteria and deters insects. The presence of this compound in the plant could explain why it could be used to treat pain, kill germs and provide people with some protection from being bitten by insects.

Another compound called rosmarinic acid has anti-inflammatory and antioxidant activity and these activities could contribute too many of the medicinal properties of holy basil. The plant also contains ursolic acid a compound that has been shown to provide some protection to enzymes in the liver that deal with the breakdown of fat in our diet. This is important as patients with diabetes often have high levels of cholesterol in their blood. The levels have been reported to decrease after taking holy basil.

1.15 Medicinal values of the Tulsi:

The tulsi or holy basil is an important symbol in the Hindu religious tradition and is worshipped in the morning and evening by Hindus at large. The holy basil is also a herbal remedy for a lot of common ailments. Here’re top fifteen medicinal uses of tulsi.

1. Healing Power: The tulsi plant has many medicinal properties. The leaves are a nerve tonic and also sharpen memory. They promote the removal of the catarrhal matter and phlegm from the bronchial tube. The leaves strengthen the stomach and induce copious perspiration. The seed of the plant are mucilaginous.

2. Fever & Common Cold: The leaves of basil are specific for many fevers. During the rainy season, when malaria and dengue fever are widely prevalent, tender leaves, boiled with tea, act as preventive against theses diseases.

In case of acute fevers, a decoction of the leaves boiled with powdered cardamom in half a liter of water and mixed with sugar and milk brings down the temperature. The juice of tulsi leaves can be used to bring down fever. Extract of tulsi leaves in fresh water should be given every 2 to 3 hours. In between one can keep giving sips of cold water. In children, it is every effective in bringing down the temperature.

3. Coughs: Tulsi is an important constituent of many Ayurvedic cough syrups and expectorants. It helps to mobilize mucus in bronchitis and asthma. Chewing tulsi leaves relieves cold and flu.

4. Sore Throat: Water boiled with basil leaves can be taken as drink in case of sore throat. This water can also be used as a gargle.

5. Respiratory Disorder: The herb is useful in the treatment of respiratory system disorder. A decoction of the leaves, with honey and ginger is an effective remedy for bronchitis, asthma, influenza, cough and cold. A decoction of the leaves, cloves and common salt also gives immediate relief in case of influenza. They should be boiled in half a liter of water till only half the water is left and add then taken.

6. Kidney Stone: Basil has strengthening effect on the kidney. In case of renal stone the juice of basil leaves and honey, if taken regularly for 6 months it will expel them via the urinary tract.

7. Heart Disorder: Basil has a beneficial effect in cardiac disease and the weakness resulting from them. It reduces the level of blood cholesterol.

8. Children’s Ailments: Common pediatric problems like cough cold, fever, diarrhea and vomiting respond favorably to the juice of basil leaves. If pustules of chicken pox delay their appearance, basil leaves taken with saffron will hasten them.

9. Stress: Basil leaves are regarded as an ‘adaptogen’ or anti-stress agent. Recent studies have shown that the leaves afford significant protection against stress. Even healthy persons can chew 12 leaves of basil, twice a day, to prevent stress. It purifies blood and helps prevent several common elements.

10. Mouth Infections: The leaves are quit effective for the ulcer and infections in the mouth. A few leaves chewed will cure these conditions.

11. Insect Bites: The herb is a prophylactic or preventive and curative for insect stings or bites. A teaspoonful of the juice of the leaves is taken and is repeated after a few hours. Fresh juice must also be applied to the affected parts. A paste of fresh roots is also effective in case of bites of insects and leeches.

12. Skin Disorders: Applied locally, basil juice is beneficial in the treatment of ringworm and other skin diseases. It has also been tried successfully by some naturopaths in the treatment of leucoderma.

13. Teeth Disorder: The herb is useful in teeth disorders. Its leaves, dried in the sun and powdered, can be used for brushing teeth. It can also be mixed with mustered oil to make a paste and used as toothpaste. This is very good for maintaining dental health, counteracting bad breath and for massaging the gums. It is also useful in pyorrhea and other teeth disorders.

14. Headaches: Basil makes a good medicine for headache. A decoction of the leaves can be given for this disorder. Pounded leaves mixed with sandalwood paste can also be applied on the forehead for getting relief from heat, headache, and for providing coolness in general.

15. Eye Disorders: Basil juice is an effective remedy for sore eyes and night-blindness, which is generally caused by deficiency of vitamin A. Two drops of black basil juice are put into the eyes daily at bedtime.

1.16 Safety

Holy basil has a long history of safe use in India. Application to the skin can cause reactions in sensitive people.

1.17 Chemical investigation of the genus Ocimum sanctum(Literature review) :

3. EXPERIMENTAL

3.1 Investigation On Ocimum Sanctum

3.2 Collection Of Plants

The plant Ocimum Sanctum was collected from Gazipur area and was identified from the department of Botany, Dhaka University. The collected fresh plants were cleaned thoroughly. The plant materials (leaves and stems) were separated from its roots and dried under mild sunlight and then at 40 ^oC in an oven. Afterwards the plants were powdered in a grinding machine (~200 smeshes). The powder was used throughout the investigation.

3.3 Phytochemical Screening Of The Plants

Chemical tests were carried out on the aqueous extract and on the powdered specimens using standard procedures to identify the constituents .

3.4 Qualitative Determination

Test For Tannins

The dried powder samples (~0.5 g) was boiled in water (20 ml) in a test tube and then filtered. A few drops of ferric chloride solution (0.1%w/v) were added and brownish green or a blue-black coloration was observed.

Test For Phlobatannins

Deposition of a red precipitate (on boiling) extract of each plant sample was boiled with aqueous hydrochloric acid (1% w/v) indicated the presence of phlobatannins.

Test For Saponin

The powdered sample (~2g) was boiled in distilled water (20 ml) in a water bath and filtered. The filtrate (10 ml) was mixed with distilled water (5 ml) and shaken vigorously for a stable persistent froth. The frothing was mixed with olive oil (3 drops) and shaken vigorously, and then the formation of emulsion was observed.

Test For Flavonoids

Three methods were used to determine the presence of flavonoids in the plant sample-

a) Dilute ammonia solution (5 ml) was added to a portion of the aqueous filtrate of each plant extract followed by addition of concentratedH₂SO₄.

b) Few drops of aluminium ion (Al³⁺) solution (1%) were added to a portion of each filtrate. A yellow colouration each observed indicating the prsence of flavonoids.

c) A portion of the powdered plant sample each case was heated with ethyl acetate (10 ml) over a steam bath for 3 min. The mixture was filtered and the filtrate (4ml) was shaken with dilute ammonia solution (1 ml). A yellow colouration was observed indicating a positive test for flavonoids.

Test For Steroids

Acetic anhydride (2 ml) was added to ethanolic extract (0.5 g) of each sample with H₂SO₄ acid (2 ml). The colour changed from violet to blue or green in some samples indicating the presence of steroids.

Test For Terpenoids (Salkowski Test)

Each extract (5 ml) was mixed in chloroform (2 ml), and concentrated H₂SO₄ acid (3 ml) was carefully added to form a layer. A reddish brown colouration at the interface was formed to show positive test for the presence of terpenoids.

Test For Cardiac Glycosides (Keller-Killani Test)

Each extracted (5 ml) was treated with glacial acetic acid (2 ml) containing ferric chloride solution (1 drop). This was underlayed with concentrated H₂SO₄ acid (1 ml). A brown ring at the interface indicates a deoxysugar characteristic of cardinolides. A violet ring may appear bellow the brown ring, while in the acetic acid layer, greenish ring may from just gradually throughout thin layer.

Table-3.1: Qualitative analysis of the phytochemical of the medicinal plants

Plants	Alkaloid	Tannin	Saponin	Flavonoid	Steroid	Terpenoid	Cardiac glycoside
Ocimum Sanctum	+	+	+	+	+	+	+

3.5 Quantitative Determination Of The Chemical Constituents

Alkaloid Determination ²⁰

Each sample (0.5 g) was taken into a conical flask (100 ml) and acetic acid (20 ml in 10%) ethanol was added. It was covered and allowed to stand for 12 hours. This was filtered and the extract was concentrated on water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to stand and the precipitate was collected and washed with dilute ammonium hydroxide solution and then filtered. The residue is the alkaloid, which was dried and weighed.

Flavonoid Determination ²²

The plant sample (1 g) was extracted repeatedly with aqueous methanol (20 ml in 80%) (CH₃OH) at room temperature. The whole solution was filtered through whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath ansd weighed to a constant weight.

Saponin Determination ²³

Each of the powder samples (5 g) were put into a conical flask and 20% aqueous ethanol (25 ml) was added. The samples were heated over a hot water bath for 4 hours with continuous stirring at about55^oC. The mixture was filtered and the residue re-extracted with 20% aqueous ethanol (50 ml). The combined extracts were reduced to 10 ml over water bath at about 90^oC. The concentrated extract (was transferred into a separatory funnel and diethyl ether (5 ml) was added and shaken vigorously. The aqueous layer was recovered while the whether layer discarded. The purification process was repeated. n-butanol (15 ml) wad added and n-butanol extracts were washed twice with aqueous 5% NaCl (3 ml of) solution). The remaining solution was heated on a water bath. After evaporation the samples were dried in an oven to a constant weight; the saponin content calculated as percentage. .

Table-3.2: Amount (% on dry powder basis) of crude alkaloid, flavonoid and saponin on the medicinal plants investigated.

Plants	Alkaloids (%)	Flavonoids (%)	Saponin (%)
Ocimum Sanctum	0.8	14.06	8.5

3.6 Extraction And Isolation Of Compounds From Ocimum Sanctum

Plant powder was taken in a few precleaned cloth thimbles. The thimbles containing the powder were placed in a Soxhlet apparatus.

The plant powder extracted separately and exhaustively in Soxhlet apparatus first with Petroleum ether (boiling point 40-60 ^oC and 60-80 ^oC) followed by hexane. All the extracts were filtered individually; the filtrate was combined together and then concentrated in a “Buchi Rotavapor” under reduced pressure. This was shown in scheme-1

EXTRACTION SHEME OF OCIMUM SANCTUM

Powder (80g)

Extraction with pet-ether (bp.40-60^oC)

Petroleum ether exract Residue

Evaporated to dryness Extracted with hexane

Gummy mass Hexane extract Residue

Evaporated to dryness

Dry mass of hexane extract (18 g)

Scheme 1: Extraction scheme of Ocimum Sanctum

3.7 Investigation Of Hexane Extract

3.8 Thin Layer Chromatography (TLC) Study

TLC analysis of the hexane extract showed the presence of one spot (Petroleum ether: Dichloromethane = 90: 20) having light brown under iodine vapor. Furthermore, TLC study showed the presence of two spots on spraying with vanillin sulfuric acid followed by heating in oven for 15 minutes. Again three spots (Dichloromethane: Ethyl acetate = 80: 20) were visible on spraying vanillin sulfuric acid followed by heating in an oven for 15 minutes. Among these three spots, two were pink and one was violet. The presence of the pink colored spot was thought to be due to the presence of steroidal or fatty acid material or both of them. It gave also positive test for steroid²³.

3.9 Fractionated By Vaccum Liquid Chromatography (VLC)

The hexane extract was concentrated to dry mass about 18g by a ‘Buchi Rotavapor’. The concentrated extract was mixed with TLC grade silica gel (60 GF₂₅₄). The mixture was made as powder for using in column. Then the sample was placed on the top of the bed of silica gel packed column (VLC). The column was then eluted with petroleum ether followed by mixtures of pet-ether and dichloromethane of increasing polarity. The elutes were collected in test tube with an about of about 20mL in each. Solvent system used as mobile phase in the analysis of hexane extract was listed in Table.

Table- Number of fractions collected in test tubes from VLC of hexane extract by using different solvent system

Solvent system

Amounts

of solvents

(mL)Number of test tubesPet- ether (%)DCM

(%)Ethyl acetate

(%)Methanol%1000003001-129820010013-169640010017-199280010020-2388120010024-2684160010027-3180200010032-3575250010036-3970300010040-4365350010044-4760400010048-5255450010053-5550500010056-5845550010059-6140600010062-6435650010065-6730700040068-8225750030083-9120800030092-100158500100101-103108000100104-10659500200107-11229800200113-118010000100119-121

3.10 Screening Of The Fractions

TLC monitored each of the fractions and the fractions of similar behaviors were combined together and marked as F₁, F₂, F₃, F₄, F₅, F₆, F₇, . This is given in Table

Table-: Screening of the fractions by the similar TLC pattern

No of test tubes	Fractions	Solvent system for TLC	TLC pattern
1-12	F1	90:10 (PE:DCM)	One spot
17-19	F2	90:10 (PE:DCM)	One spot with tailing
32-35	F3	70:30 (PE:DCM)	Two spots with tailing
62-64	F4	20:80 (DCM:PE)	One spot
92-100	F5	20:80 (PE:DCM)	One spot with tailing
101-103	F6	20:80 (PE:DCM)	no spots
119-121	F7	20:80 (EA:DCM)	two spots with tailing

3.11 Attempt Of Purification And Characterization of the Fractions

A