INTRODUCTION:

Introduction to Herbal Medicine:

The word herb, as used in herbal medicine, also known as botanical medicine or, in Europe, as Phytotherapy or Phytomedicine means a plant or plant part that is used to make medicine to assist the healing process during illness and disease. An herb can be a leaf, a stem, a root, a seed, a fruit, a flower, or bark, used for its medicinal properties. These may be used in many forms, e.g. fresh, dried, cut, as a powder, ointment, tincture, or oil extract, or made into liquid by infusion or decoction.

Herbs have provided all living organisms with medicine from the earliest beginnings of civilization. Throughout history, various cultures have handed down their accumulated knowledge of the medicinal use of herbs. About 25% of the modern drugs currently used by doctors have a component of an herb. ^[1]

Plants have evolved the ability to synthesize chemical compounds that help them defend against attack from a wide variety of predators such as insects, fungi and herbivorous mammals. By chance some of these compounds whilst being toxic to plant predators turn out to have beneficial effects when used to treat human diseases. Such secondary metabolites are highly varied in structure many are aromatic substances, most of which are phenols or their oxygen-substituted derivatives.

It has been estimated that an average of 500,000 plant species on earth today, with the number varying depending on whether subspecies are included. Approximately 5,000 of these have been studied at length by modern science for their medicinal qualities.

All parts of a tree or plant absorb the nutrients from the soil dissolved minerals and liquid material and pass them on from the root through the stem bark to the leaves, fruits, and flowers by transpirational pull. Root bark may have different properties than the stem bark or the leaves. Most believe that the roots extract the most potent chemicals from the soil, mainly minerals, and as these are passed on through the tree/plant bark they get less and less concentrated as they reach the leaves where a chemical change occurs through insolation the photo effects from the sun. In the leaves photosynthesis takes place and this process creates change and the difference between the chemicals components derived from the leaves compared with those in the tree bark or the root bark, which is protected from this exposure. But this is not the rule in every case. On the contrary, there are certain plants where the essential curative properties are in the leaf, flower, or seed. An experienced herbalist knows this information from extensive experience, research and experiments. Medicinal plants contain varying degrees of chemicals and they have a direct impact on physiological active. Herbal medicine is useful all over the world. ^[2]

Importance of herbal medicine:

Ø The usage of herbs to treat a variety of different ailments is universal, and exists in every human culture on Earth. Despite this, the largest use of medical herbs still occurs in societies which are not fully industrialized.

Ø Because of the high costs involved with manufacturing modern medicines, many people living in developing nations simply do not have the financial resources to pay for them, and as a result, they are forced to use natural herbs as an affordable alternative.

Ø In recent years, many people living in industrialized countries have begin taking a second look at a herbal medicines due to the rising cost of medicine and healthcare in their own nations

Ø The alternative health industry has become a billion dollar a year business, and this is driving the quest to find new plants or chemicals which are useful. A number of statistics indicate that over 20% of all medicines produced in the United States have been taken from plant.^[3]

Description of Ziziphus maurtiana:

Biological source: It is obtain from the root and fruit of Ziziphus maurtiana belonging to family Rhamnaceae. Small tree with prickly thorn-like stipules and small simple alternate 3-nerved leaves white (or tawny) tomentose beneath, dark green above, ovate or subelliptic, or slightly rhomboid, mostly less than 6 cm long, finely serrulate, flowers bisexual or male, greenish, in small cymes to 10 cm long; about 5 mm wide; calyx 5-toothed; petals 5; disc large; ovary with 2 styles; fruit a drupe with sweet sour mealy flesh and a 2-seeded stone, orange when ripe".

Propagation:

"Seed is spread by birds native animals, stock, feral pigs and humans who eat the fruit and expel the seeds. Spread also by international plantings as a fruit tree"

Tree of Ziziphus maurtiana

Chemical constituents

It is a rich source of cyclopeptide alkaloids, lupane and triterpenes . Cyclopeptidmacrocycles of Ziziphus species showed interesting biological properties, including, for example, sedative, analgesic, antibacterial, antifungal and, antiplasmodial activity etc .

MATERIALS AND METHODS:

Collection and Authentication of Plant Material:

The plant of Ziziphus maurtiana were collected during the month of October from Deer park Moradabad and authenticated by Dr. D.V. Amla (Scientist G ) sample no SOP-202 at National Botanical Research Institute, Lucknow.

Methods: As given below

Ø Macro and Microscopical characters

Ø Physiochemical parameters

Ø Loss on drying

Ø Antimicrobial activity

1. Pharmacognostical Study: ^[4,6,8]

1.a Macroscopical Characters:

Table 1:

1.	Size	Length 10-15 cm, Diameter 4-5 cm
2.	Colour	Brownish
3.	Surface Character	Rough
4.	Shape	Cylindrical
5.	Fracture	Fibrous
6.	Odour	Odourless
7.	Taste	Bitter

1.b Microscopical Characteristics:

Epiblema:

This is the single, outermost layer of thick walled cells. The outer walls of most of these cells extend outwards and form unicellular root hairs. This layer is used for absorption of water and solutes form the soil and, therefore, it has no cuticle. Root hairs increase the absorbing surface of the root.

Cortex:

This consists of many layers of unlignified, oval shaped parenchymatous cells with small intercellular spaces between them, some cells contain prism of calcium oxalate crystals.

Endodermis:

This is a single, ring-like layer of barrel –shaped cells which are closely packed with-out intercellular spaces. The endodermis is the inner most layer of the cortex and occurs as a ring around the stele.

Conjunctive Tissue:

The Parenchyma lying between the xylem and phloem bundles constitutes the conjunctive tissue

Vascular bundles:

These are always arranged in a ring and are radial i.e., xylem and phloem are situated at different radii. The protoxylem is always away from the centre and metaxylem towards the centre. This condition of xylem is called exarch. The vascular bundles are tri-arch.

1.c Powder Characteristics: ^[7,9,10]

Table 2: Staining

S.No.	Reagents	Observation	Characteristics
1.	Phloroglucinol + Conc. HCl (1:1)	Pink	Xylem vessels, Xylem fibers, Trichomes
2.	Iodine solution	Blue	Starch
3.	Acetic acid	Insoluble	Calcium oxalate crystals
4	Hydrochloric acid	Soluble	Calcium oxalate crystals
5	Sulphuric acid(60% w/w)	Soluble, needle of calcium sulphate on standing	Calcium oxalate crystals

2. Physicochemical property:

2.a Determination of ash:

Ash consists of inorganic radicals like phosphates, carbonates and silicates of sodium, potassium; calcium etc. is used to determine quality and purity of a crude drug.

The ash remaining following ignition of medicinal plant material is determined by different methods which measure total ash, acid insoluble ash, water soluble ash and sulphated ash.

The total ash method is designed to measure the total amount of material remaining after ignition. This includes both “physiological ash”, which is derived from the plant tissue itself. And “non-physiological ash”, which is the residue of the extraneous matter (e,g sand and soil) adhering to the plant surface.

Procedures:

For determination of different ash values, the root of Ziziphus maurtiana was powdered. The powder was passed through sieve no. 40 and used as follows:-

Total ash:

1 g air dried powder was placed in a previously ignited and tarred crucidly. The powder was spreaded in an even layer and ignited by gradually increasing the heat to 500 – 600 ⁰C until it is white, indicating the absence of carbon. Then crucible was cooled in desiccators. The ash was weighed and percentage of total ash with reference to air dried powder was calculated. The total ash value is given in table no 3.

Acid- insoluble ash:

25 ml of hydrochloric acid (70g/l) was added to the crucible containing the total ash and boiled gently for 5 minutes. The insoluble matter was collected on the ash less filter paper and washed with hot water until the filtrate is neutral. The filter paper was transferred to the original crucible and ignited to a constant weight. The residue was to cool in a suitable dessicator for 30 min. The ash was weighed without delay and percentage of acid-insoluble ash with reference to air dried powder was calculated. The acid-insoluble ash is given table no 3.

Water soluble ash:

25ml of water was added to the crucible containing the total ash and boiled for 5 min. The insoluble matter was collected on the ash less filter paper and washed with hot water.

The filter paper was transferred to the original crucible and ignited to a constant weight at a temperature not exceeding 450^oC. The residue was allowed to cool in a suitable desiccator for 30 min. The weight of the residue in milligram was subtracted from the weight of total ash. The ash was weighed without delay and percentage of water soluble ash with reference to air dried powder was calculated. Water soluble ash value is given in table no.

Table 3: Ash values

S.no.	Analytical Parameters	*Ash value (% w/w)
1.	Total Ash	6.5%
2.	Acid Insoluble ash	2.0%
3.	Water Soluble ash	5.5%

*Average of three determinations

2.b Determination of extractable matter:

This method determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for material for which as yet no suitable chemical or biological assay exists.

Method:

Cold Maceration:-

The root of Ziziphus maurtiana was powdered. The powder was passed through sieve no. 20and used as follows:-

Alcohol –soluble extractive:-

5 g of air dried coarse powder was macerated with 100 ml of ethanol of the specified strength in a closed flask for 24 hours, shaken frequently during the first 6 hours and allowed to stand for 18 hours. Therefore filtered rapidly taking precaution against loss of ethanol, evaporated 25 ml of the filtrate to dryness in a tarred fiat- bottomed shallow dish, dried at 105^oc and weighed .The percentage of ethanol- soluble extractive was calculated with reference to the air dried drug. The ethanol-soluble extractive value is given in table no 4.

Water-soluble extractive:-

Proceed as directed for the determination of ethanol –soluble extractive, using chloroform water instead of ethanol. The water-soluble extractive value given in table no 4.

Petroleum ether- soluble extractive:-

Proceed as directed for the determination of ethanol –soluble extractive, using petroleum ether instead of ethanol. The petroleum ether- soluble extractive value is given in table no 4.

Table: 4 Extractive Values:-

S. No.	Analytical parameter	Extractive value (% w/w)*
1.	Petroleum ether-soluble extractive	0.32%
2.	Alcohol-soluble extractive	2%
3.	Water- soluble extractive	52%

*Average of three determination

3. Loss on drying:

Loss on drying is the loss of weight expressed as percentage w/w resulting from water and volatile matter of any kind that can be driven off under specified conditions. The test is carried out on a well mixed sample of substance is in the form of crystals, reduce the size by rapid crushing to a powder.

An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects and deterioration following hydrolysis. Limits for water content should therefore be set for every given plant material. This is especially important for materials that absorb moisture easily or deteriorate quickly in the presence of water.

Procedure:-

1g of dried powder was accurately weighed andplaced in a previously dried weighing bottle. The sample was dried in a oven at 100-105⁰c until two consecutive weighing do not differ by more than 5 mg. The loss of weight in mg per of air dried material was calculated.

Report:

The loss on drying was found to be 16% w/w.

4. Antibacterial activity: ^[5,11,12]

The present study was undertaken to determine antibacterial activity of ethanolic extract of Ziziphus maurtiana. The powdered root of Z. maurtiana was extracted with ethanol. Antibacterial activity of ethanol extract was determined against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis by disc diffusion method.

Bacterial strains

Bacillus subtalis, Staphylococus aureus, E. coli and Psudomonas aeruginosa strains used in this study.

Solvent Extraction:

Extracted successively with ethanol solvent in Soxhlet extractor for 48h. The solvent extracts were concentrated under reduced pressure and preserved at 5°C in airtight bottle until further used.

Maintenance of Test Microorganism for Antimicrobial Studies:

The bacteria were maintained on nutrient broth (NB) at 37°C

Formula (Nutrient broth):

Peptone: 5gm/ltr

Beef Extract: 1.5gm/lit

Yeast Extract: 1.5gm /lit

Agar: 15gm/lit

Preparation of Inoculum: The gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative bacteria (Escherichia coli, Pseudomonas were pre-cultured in nutrient broth.

Anti-bacterial Activity: The root extract of Ziziphus mauritian, was tested by the disc diffusion method [10]. Different concentration of the extracts was prepared by reconstituting with ethanol.

Disc Diffusion Method:

Introduction:

When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar .This diffusion will place the chemical in the agar only around the disc .The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on agar it will not grown in the area around the disc is known as zone of inhibition.

Principle:

Antiseptics, disinfectants and antibiotics are used in different ways to combat microbial growth. Antiseptics are used on living tissue to remove pathogens. Disinfectants are similar in use but are used on inanimate objects. Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria. Many antibiotics are chemically altered to reduce toxicity, increase solubility, or give them some other desirable characteristic that they lack in their natural form. Other substances have been developed from plants or dyes and are used like antibiotics. A better term for these substances is antimicrobials, but the term antibiotic is widely used to mean all types of antimicrobial chemotherapy.

The amount of organism used is standardized using a turbidity standard. This may be a visual approximation using a McFarland standard 0.5 or turbidity may be determined by using a spectrophotometer (optical density of 1.0 at 600 nm). For antibiotic susceptibility testing the antibiotic concentrations are predetermined and commercially available. Each test method has a prescribed media to be used and incubation is to be at 35-37o C in ambient air for 18-24 hours. The disc diffusion method for antibiotic susceptibility testing is the Kirby-Bauer method. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method.

This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. The standardized method for antiseptic and disinfectant testing is more rigorous and more difficult to reproduce in a student laboratory. Two common tests are the Phenol Coefficient Test (a comparison of the effect of the chemical and phenol on several organisms) and the Use Dilution Test (testing the chemical under actual conditions of use). A disc diffusion test can be used to approximate the Use Dilution Test. The chemical under consideration is used to saturate a filter paper disc. This disc is then used to introduce the chemical to the agar for testing. The actual zone sizes have not been standardized as in the Kirby-Bauer method, but a comparison of zone sizes for the same chemical among organisms will provide an approximate effectiveness of the chemical.

Procedure:

1. All the apparatus along nutrient agar media wera sterilized.

2. After sterilization taken to the laminar air flow chamber.

3. Nutrient media was poured over all the petidish.

a. One was kept as a contol.

b. Four kept for testing of test drug each inoculate with gram positive and gram negative bacteria.

c. Two kept testing of standard drug each inoculate with gram positive and gram negative bacteria.

4. Now 6 mm diameter width wafers were applied with drug solution with volume of 4 µl and 6 µl, such that two of each concentration placed on each petridisc with E. coli and bacteria strain which were kept for test drugs.

5. The solutions were poured by using micropippete after sterilization.

6. A standard ciprofloxacin tablet was placed on each petridisc with strain.

7. Control petridisc was rendered free of any microorganism and drug.

8. These petridish were transferred to incubator for 24 hrs maintaining at 37⁰C growth were observed on each petridisc.

9. After observation the petridish were again kept in autoclave for discarding the material.

10. The material in petridisc was discarded on news paper thrown in dustbin.

RESULTS AND DISCUSSION:

The plants of Ziziphus maurtiana. were collected from Ranikhet regions and authenticated for my project work. The present investigation deals with Pharmacognostical studies of root of this plant.

Except few biological studies, not much work has been done on this plant. Therefore, It has been proposed to undertake the present work entitled ‘’Pharmacognostical Studies on root of Ziziphus maurtiana.’’

The macroscopic characters of root including the length 4-8 cm and thickness 4-20 mm, cylindrical, curved, branched, brown in color, aromatic odour and bitter in taste.

In microscopic studies, the transverse sections of root showed few light brown colored cork layer of regularly arranged parenchymetous cells; isodiametric, thin walled parenchymetous cells containing starch grains’ bicollateral vascular bundle is present.

Powder microscopy showed the presence of calcium oxalate crystals of average length of about 15.98 µ and 12.24µ wide, Simple and rounded starch grains are present. Covering trichomes are uniseriate, bent, acute apex and unicellular in nature.

In physicochemical studies, different parameters like total ash, water-soluble, acid-insoluble 12%, 3%, 1.5%, w/w respectively; extractive values like petroleum ether, ethanol and water 2.4%, 3%, 4% w/w respectively and loss on drying 11% w/w were observed.

Preliminary phytochemical analysis of aqueous and alcoholic extract was carried out for detection of different compounds. Both the extracts were found to be positive for the presence of alkaloids, glycosides (cardiac and flavenoids), protein, amino acids and volatile oils.

The anti microbial activity of Ziziphus maurtiana show following result –

Name of the bacteria	Mean diameter of zone of inhibition						Control
	Ethanolic extract of root of Ziziphus maurtiana				Ciprofloxacin Tablet (30µg/disc)	Ampicillin (25 µg/disc)
	Volume
	10µl	1 µl	0.1 µl	0.01 µl
E.coli	10 mm	6mm	12mm	7mm	15mm	19mm	Nill
Bacillus subtalis	7 mm	4mm	8mm	6mm	10 mm	16mm	Nill
Pseudomonas aeruginosa	8 mm	4mm	11mm	5mm	17mm	21mm	Nill
Staphylococus aureus	10 mm	6mm	10mm	8mm	14mm	24mm	Nill

1.c Powder Characteristics: [7,9,10]

1.c Powder Characteristics: ^[7,9,10]