Studies on Chebulinic Acid Extraction from Terminalia chebula species

CONTENTS

Abstract

Introduction

Literature Review

Materials and Methods
Extraction and Processing
Quantative Determination of Phytoconstituents
Optimization of Physico-Chemical parameters
Soxhlet extractor- An experimental & modelling studies
Fermentation
Pratition coefficient
Column chromatography
Anticancer activity

Results and Discussions
Extraction and Processing
Quantative Determination of Phytoconstituents
Optimization of Physico-Chemical parameters
Soxhlet extractor- An experimental &modelling studies
Fermentation
Pratition coefficient
Column chromatography
Anticancer activity

Conclusion

References

Abbrevations

Appendix A – List of Figures

Appendix B – List of Tables

Appendix C – List of Graphs

List of Conference Presentations done during project

List of Journal Publications done during project

ABSTRACT

Herbal drug product has a special place in the world of pharmaceuticals. Terminalia chebula is a deciduous tree, used in traditional medicines. It is reported to contain various bio chemical compounds such as tannins, chebulinic acid, ellagic acid, gallic acid, punicalagin, flavonoids etc. It has been reported as antioxidant, antidiabetic, antibacterial, antiviral, antifungal, anticancerous, antiulcer, antimutagenic, wound healing activities etc.

The optimization of physico-chemical parameters like effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with ethanol and methanol as solvents and pH for the extraction of Total Phenolic Content, Chebulinic acid and Quercetin were studied. For the extraction of Total Phenolic Content, the optimum results were observed for the effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with ethanol as solvent and pH were ethanol, 1 day, 1hrs, 125 microns, 50% (v/v), 1:1 ratio and 7.0 respectively. The highest Total Phenolic Content concentration for optimized conditions was 2.25µg/dl. For the extraction of Chebulinic acid, the optimum results were observed for the effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with ethanol as solvent and pH were ethanol, 1 day, 1hrs, 125 microns, 50% (v/v), 1:1 ratio and 7.0 respectively. The highest Chebulinic acid concentration for optimized conditions was 3.4mg/ml. For the extraction of Quercetin, the optimum results were observed for the effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with methanol as solvent and pH were methanol, 2 days, 1hrs, 125 microns, 60% (v/v), 1:1 ratio and 6.0 respectively. The highest Quercetin concentration for optimized conditions was 0.54µg/cl.

The extraction was carried out by employing various organic solvents using Soxhlet extractor method. Ethanol was found to be the best solvent for the extraction of Chebulinic acid from Terminalia chebula species. Soxhlet extractor was carried out using ethanol at different extraction times to verify the mathematical model proposed in this work. The final form of the proposed models were Es = 0.1650 (1 - e -0.0415t) for Chebulinic acid where Es = yield extract (grams of Chebulinic acid per gram of dried sample) and t = extraction time (min) and Es = 0.925(1 - e -0.0499t) for Total phenolic content where Es = yield extract (mg of Total phenolic content per gram of dried sample) and t = extraction time (min). The model showed good agreement with the experimental data by generating Average absolute relative deviation (AARD) of about 0.1176 ± 20.80% for Chebulinic acid and 0.7169 ± 16.06% for Total phenolic content.

The optimization of physico-chemical parameters required for obtaining the highest production of Chebulinic acid using Baker’s yeast (Saccharomyces cerevisiae). From the fermentation process, the incubation period, pH, biomass concentration, partition coefficient for the aqueous extract were 142 hr, 4.2, 0.3281gm/ml, 1.70. Similarly for the 50% (v/v) ethanolic extract were 192 hr, 4.3, 0.3926gm/ml, 3.83 and for the hexane extract were 264 hr, 4.3, 0.3671gm/ml, 2.30. It was concluded that 50% (v/v) ethanolic extract showed best results for the highest Chebulinic acid production and found to be 8.6 mg/ml. Similarly the concentration was 4.2mg/ml from the aqueous extract and the concentration was 7.5mg/ml from hexane extract.

Among the extraction process (Batch, Soxhlet & Fermentation process) the highest Chebulinic acid production was obtained from fermentation process. From the batch process the chebulinic acid concentration was observed to be 3.4mg/ml at 60 min and the concentration was increased to 6.6mg/ml at 75 min from soxhlet extraction. Similarly the chebulinic acid concentration was increased to 8.6mg/ml at 192 hrs from fermentation process. The partition coefficient for fermentation, soxhlet & batch extraction were found to be 3.83, 2.0 & 0.64.

The extraction of Chebulinic acid from Terminalia chebula by Soxhlet extraction and purify the Chebulinic acid by using Column chromatography. From the Soxhlet extraction the Chebulinic acid concentration was 6.6 mg/ml and it was increased to 9.4mg/ml from the Column chromatography. The purity of Chebulinic acid was improved by Column chromatography.

Chebulinic acid showed many bioactivities including inhibition of cancer cell growth, inhibiting the contractile responses of cardiovascular muscles, anti-fungal, anti-bacterial activities etc. In-vitro anti-cancer activity of Chebulinic acid on Colon adenocarcinoma HT-29 cancer cell lines by using MTT cell growth inhibition assay was stuied. The maximum percentage inhibition of cancer cell lines for Chebulinic acid was found to be 41.2% at a dose of 200µg/ml. Finally the Chebulinic acid extraction from Terminalia chebula plays a vital role in medicine, biotechnology and various pharmacological activities.

INTRODUCTION

Biotechnology is the application of living organisms and their components to industrial products and process. It is based on biology, especially when used in agriculture, food science, and medicine. The history ofbiotechnology begins with zymotechnology, which commenced with a focus onbrewing techniques for industrial fermentation, particularly beer. In 1919, Karl Ereky, a Hungarian engineer, coined the term Biotechnology to describe the interaction of biology and human technology. He envisioned a new era of technology based on using biology to turn raw materials into socially useful products. After the First World War, as "biotechnology" entered German dictionaries and was taken up abroad by business-hungry private consultancies as far away as the United States. In Chicago, at the end of World War I encouraged biological industries to create opportunities for new fermentation products, in particular a market for non-alcoholic drinks.The belief that the needs of an industrial society could be met by fermenting agricultural waste was an important ingredient of the "chemurgic movement". Fermentation-based processes generated products of ever-growing utility. In the 1940s,penicillinwas the most dramatic. While it was discovered in England, it was produced industrially in the U.S. using a deep fermentation process. The origins of biotechnology culminated with the birth ofgenetic engineering. There were two key events that have come to be seen as scientific break throughs beginning the era that would unite genetics with biotechnology. One was the 1953 discovery of the structure of DNA by Watson and Crick, and the other was the 1973 discovery by Cohen and Boyer of a recombinant DNA technique by which a section of DNA was cut from the plasmid of an E. coli bacterium and transferred into the DNA of another.This approach could, in principle, enable bacteria to adopt the genes and produce proteins of other organisms, including humans. Popularly referred to as "genetic engineering," it came to be defined as the basis of new biotechnology. In 1981 the European federation of biotechnology defined as “Integrated use of Biochemistry, Microbiology and Chemical engineering in order to achieve the technological application of microbs and cultured tissue cells”. Defining the scope of biotechnology is not easy because it overlaps with so many industries, such as the chemical industry or food industry being majors, but biotechnology has found many applications in pharmacology and pharmacognacy.

Biotechnology is a field of biology that involves the use of living things in engineering, technology, medicine etc. Modern use of the terms refers to genetic engineering as well as tissue culture technologies. However, the concept encompasses a wider range and history of procedures for modifying living organism according to human purposes, going back to domestication of animals, cultivation of plants and “improvements” to these through breeding programs that employ artificial selection and hybridization. By comparison to biotechnology, bioengineering is generally thought of as a related field with its emphasis more on mechanical and higher systems approaches to interfacing with and exploiting living things. The United Nations Convention on Biological Diversity defines biotechnology as: "Biotechnology means anytechnological application that uses biological systems, living organisms, or derivatives thereof, to make or modify productsor processes for specific use."

Applications

There are numerous applications for biotechnologies, and the numbers of these applications are constantly increasing. Many sectors of agriculture, industry and services use biotechnologies for various purposes:

- To produce substances with special characteristics (for example, biomedicines, additives for food stuffs, chemical products and biofuels);
- To improve traditional production processes (through bio catalysis, for example);
- To make research and development into new products easier (innovative medicines for example);
- To influence the breeding and the propagation of plants and animals;
- To provide services (diagnostic activities, reclamation of polluted areas, for example).

1. Fermentation

This is one the very first applications of food biotechnology and contains several different types, which utilizes bacteria to digest certain carbohydrate sources and produce alcohol, acid, carbon dioxide, and amino acids/peptides. The use of fermentation can generate numerous food products such as wine, beer, cheese, bread, and yog hurt etc.

2. Genetically Modified Food This application of food biotechnology is when a gene from one species is transferred into another species. Often times, the gene will have a trait that is desirable to another food species, such as a longer shelf life or preventing diseases. Eg: corn, tomato & golden rice.

3. Industrial Biotechnology

Industrial biotechnology applies the techniques of modern molecular biology to improve the efficiency and reduce the environmental impacts of industrial processes like textile, paper and pulp, and chemical manufacturing. For example, industrial biotechnology companies develop biocatalysts, such as enzymes, to synthesize chemicals. Enzymes are proteins produced by all organisms. Using biotechnology, the desired enzyme can be manufactured in commercial quantities. Commodity chemicals (e.g., polymer-grade acrylamide) and specialty chemicals can be produced using biotech applications.

Traditional chemical synthesis involves large amounts of energy and often-undesirable products, such as HCl. Using biocatalysts, the same chemicals can be produced more economically and more environmentally friendly. An example would be the substitution of protease in detergents for other cleaning compounds. Detergent proteases, which remove protein impurities, are essential components of modern detergents. They are used to break down protein, starch, and fatty acids present on items being washed. Protease production results in a biomass that in turn yields a useful by product- an organic fertilizer. Biotechnology is also used in the textile industry for the finishing of fabrics and garments. Biotechnology also produces biotech-derived cotton that is warmer, stronger, has improved dye uptake and retention, enhanced absorbency, and wrinkle- and shrink-resistance. Some agricultural crops, such as corn, can be used in place of petroleum to produce chemicals. The crop’s sugar can be fermented to acid, which can be then used as an intermediate to produce other chemical feedstocks for various products. It has been projected that 30% of the world’s chemical and fuel needs could be supplied by such renewable resources in the first half of the next century. It has been demonstrated, at test scale, that biopulping reduces the electrical energy required for wood pulping process by 30%.

4. Environmental Biotechnology

Environmental biotechnology is the used in waste treatment and pollution prevention. It can more efficiently clean up many wastes than conventional methods and greatly reduce our dependence on methods for land-based disposal. Every organism ingests nutrients to live and produces by-products as a result. Different organisms need different types of nutrients. Some bacteria thrive on the chemical components of waste products. Environmental engineers are used as bioremediation, the broadest application of environmental biotechnology, in two basic ways.

They introduce nutrients to stimulate the activity of bacteria already present in the soil at a waste site, or add new bacteria to the soil. The bacteria digest the waste at the site and turn it into harmless by products. After the bacteria consume the waste materials, they die off or return to their normal population levels in the environment. Bioremediation is an area of increasing interest. Through application of biotechnical methods, enzyme bioreactors are being developed that will pretreat some industrial waste and food waste components and allow their removal through the sewage system rather than through solid waste disposal mechanisms. Waste can also be converted to biofuel to run generators. Microbes can beinduced to produce enzymes needed to convert plant and vegetable materials into building blocks for biodegradable plastics. In some cases, the by-products of the pollution-fighting microorganisms are themselves useful. For example, methane can be derived from a form of bacteria that degrades sulfur liquor, a waste product of paper manufacturing. This methane can then be used as a fuel or in other industrial processes.

5. Animal Biotechnology

Animal biotechnology is the application of scientific and engineering principles to the processing or production of materials by animals or aquatic species to provide goods and services. Examples of animal biotechnology include generation of transgenic animals or transgenic fish (animals or fish with one or more genes introduced by human intervention), using gene knockout technology to generate animals in which a specific gene has been inactivated, production of nearly identical animals by somatic cell nuclear transfer (also referred to as clones), or production of infertile aquatic species.

Transgenics

Since the early 1980s, methods have been developed and refined to generate transgenic animals or transgenic aquatic species. For example, transgenic livestock and transgenic

aquatic species have been generated with increased growth rates, enhanced lean muscle mass, enhanced resistance to disease or improved use of dietary phosphorous to lessen the environmental impacts of animal manure. Transgenic poultry, swine, goats, and cattle also have been produced that generate large quantities of human proteins in eggs, milk, blood, or urine, with the goal of using these products as human pharmaceuticals. Examples of human pharmaceutical proteins include enzymes, clotting factors, albumin, and antibodies. The major factor limiting widespread use of transgenic animals in agricultural production systems is the relatively inefficient rate (success rate less than 10 percent) of production of transgenic animals. NIFA has supportedresearch projects to generate transgenic animals or transgenic aquatic species with enhanced production or health traits.

6. Medical Biotechnology

Medical biotechnology is an application of biotechnology that touches the lives of individuals every day. Both wellness and illness have ties to biotechnology. Advances in biology over the last 20 years have generated new insights into the causes of disease. This new level of understanding has, in turn, created opportunities for the development of new therapies, drugs, diagnostic tools and research/clinical instrumentation. Medical biotechnology is one of the fastest growing opportunities for employment in the medical research field.Scientists are looking at the genetic causes of diseases, genetic links among family members, and individualized cures. As the Human Genome Project continues to map the locations of genes on human chromosomes, more solutions to the cause, prevention and cure of diseases will be discovered.

The major areas are related to medical biotechnology

- Human Genome Project and its influence on the medical biotechnology
- Detecting Genetic Diseases
- Biotech in the Hospital

a.Pharmacogenomics b. Genetherapy c. Array

Biotechnology of Medicinal Plants

Medicinal plants are the most important source of life saving drugs for the majority of the world`s population. Plant secondary metabolites are the economically important as drug, fragrances, pigments, food additives and pesticides. The biotechnological tools are important to select, multiply, improve and analyze medicinal plants. Plant cell culture systems represent a potential renewable source of valuable medicinal compounds, flavours, fragrances and colorants, which cannot be produced by microbial cells or chemical synthesis. In-vitro production of secondary metabolites in plant cell suspension culture has been reported from various medicinal plants and bioreactors are the key step towards commercial production of secondary metabolites by plant biotechnology. Genetic transformation is a powerful tool for enhancing the productivityof novel secondary metabolites; especially by Agrobacterium tumefacians. Combinatorial biosynthesis is another approach in the generation of novel natural products and for the production of rare and expensive natural products. DNA profiling techniques like DNA microarrays save as suitable high through put tools for the simultaneous analysis of multiple genes and analysis of gene expression that becomes necessary for providing clues about regulatory mechanism, biochemical pathways and broader cellular functions.

List of Medicinal Plants

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Table 1: List of Medicinal Plants

Terminalia chebula

Terminalia chebula is a moderate tree used in traditional medicines. It is belongs to the family combretaceae. It is commonly called as Black myrobalan, Ink tree (or) Chebulic myrobalan as shown in figure 1. It is extensively used in unani, ayurveda and homeopathic medicine. Terminalia chebula is a popular traditional medicine not only used in India but also in other countries of Asia and Africa. It is called the “King of Medicines” in Tibet and is listed first in the Ayurvedic material medica because of its extraordinary power of wound healing and a wide spectrum of medicinal properties. This is used in traditional medicine due to the wide spectrum of pharmacological activities associated with the biologically active chemicals present in this plant. A numbers of chemical constituents are isolated from this plant extract. Mainly tannins, gallic acid, chebulinic acid[39], chebulic acid, chebulagic acid etc constituents are present. Terminalia chebula possesses antibacterial, antifungal, antiviral, antidiabetic, antimutagenic, antioxidant[42], antiulcer, and wound healing properties. It is used for the treatment of number of diseases like cancer, paralysis, cardio vascular diseases, ulcers, leprosy, arthritis, gout, epilepsy etc. It is used extensively in the preparation of many ayurvedic formulations for infectious diseases such as chronic ulcers, leucorrhoea, pyorrhoea and fungal infections of the skin. It is used to prevent aging and impart longevity, immunity and body resistance against disease. Terminalia Chebula is excellent for the digestive system. The fruit pulp is used as a dentifrice to cure bleeding and ulceration of gums. It has beneficial effect on all the tissues.

Classification

Kingdom - Plantae

Division - Magnoliophyta

Class - Magnoliopsida

Order - Myrtales

Family - Combretaceae

Genus - Terminalia

Species - chebula

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Figure 1: Terminalia chebula plant

(source: http://www.herbsfor.net/wp-content/uploads/2010/10/Terminalia-chebula.jpg.)

Synonyms

Blackmyrobalan

Inktree

Chebulicmyrobalan

Myrobalanuschebula

Aralu

Zhang-Qin-Ge
Myrobalane

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Urdu: Haejarad

Habitat

It grows in India, Myanmar, Bangladesh, Iran, Egypt, Turkey, China etc. In India Haritaki tree is grows in deciduous forests and found in North India and South words to the Deccan table lands at 1000 to 3000 ft. In Myanmar country grow up to 5000 ft. Its consists of pericarp of mature fruit of Terminalia chebula, a moderate sized (or) large tree found throughout India chiefly in deciduous forests and areas of light rain fall but occasionally also in slightly moist forests up to about 1500 meter elevation throughout India , flowers appear from April – August and fruits ripen from October – January.

Macroscopic characteristics

Tree: It is a deciduous tree, younger stems glabrescent, woody.

Leaves: These are 10 – 20 cm long, sub – opposite, simple; exstipulate; petiolate; laminae broadly elliptic to elliptic – oblong, rarely ovate, the bases obtuse, the margins entire, the tips acute, glabrescent. These leaves are shown in figure 2.

Inflorescence: Its paniculate spikes, terminal and axillary; peduncles tomentose; bracts subulate, small, caducous.

Flowers: In the figure 3 the flowers of terminalia chebula are shown, which are 2 mm long , 3-4 mm in diameter; bracts nearly glabrous, 1.5-2.0 mm long; calyx outside glabrous, inside densely villous, calyx-segments triangular; stamens 3-4 mm long; ovary glabrous, ovoid, 1 mm long; style glabrous, 2.5- 3.0 mm long.

Fruit: It is a drupe, glabrous, sub globose to ellipsoid, 2.5 – 5.0 cm by 1.5-2.5 cm,

usually smooth or frequently 5-angulate, ridged, wrinkled, turning blackish when dry

Fruits contain astringent substances - tannic acid, chebulinic acid, gallic acid etc. Resin and a purgative principle of the nature of anthraquinone and sennoside are also present. These fruits are shown in figure 4.

Seed: one, rough, ellipsoid, 1.0-2.0 cm by 0.2 -0.7 cm and without ridges.

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Figure 2: Leaves

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Figure 3: Flowers

(source: www.doctorayur.com) (source: www.123rf.com)

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Figure 4: Dry fruits

(source: www.herbexinternational.com)

Microscopic characteristics

Transverse section of the fruit shows epicarp composed of a layer of epidermal cells, the outer tangential wall and upper portion of the thick radial walls. Mesocarp, 2 or 3 layers of collenchymas^[16]followed by a broad zone of parenchyma with fibres and sclereids in groups and vascular bundles, scattered; fibres, simple pitted walls; porous parenchyma; sclereids, various shapes and sizes, mostly elongated; tannins and aggregate crystals of calcium oxalate in parenchyma; starch grains simple rounded or oval in shape, measuring 2-7 μm in diameter. Endocarp consists of thick walled sclereids of various shapes and sizes, mostly elongated. Fibres, sclereids and vessels lignified. Testa, one layer of large cubical cells, followed by a zone of reticulates parenchyma and vessel; tegmen consists of collapsed parenchyma. Cotyledon folded and containing aleurone grains, oil globules and some rosette aggregate crystals.

Powder: The powder of this fruit is brownish in color and is shown in figure 5.The observation under microscope shows a few fibers, vessels with simple pits and groups of sclereids.

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Fig 5: Powdered dry fruits of Terminalia chebula

(source: www.imexbb.com )

Phytochemistry

Terminalia chebula contains the triterpenes arjunglucoside 1, arjungenin and the chebulosides 1&2. Other constituents contains tannins up to 30%, chebulic acid 3-5%, chebulinic acid 30%, chebulagic acid[55], tannic acid 20-40%, ellagic acid, 2,4-chebulyi–β-D-glucopyranose, gallic acid[26], ethyl gallate, punicalaginter flavin A , terchebin, some purgative of the nature of anthraquinone , amino acids, alkaloids, glycosides, sterols likeβ-sitosterol and stigmasterol, flavonoids like luteolin, rutins, and quercetin etc. The structures of the gallic acid, chebulinic acid, chebulic acid and chebulagic acid are shown in figure 6.

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Figure 6: Structures of gallic acid, chebulinic acid, chebulic acid & chebulagic acid

(above all structures downloaded from Wikipedia)

Uses of Terminalia chebula

It is given as adjuvant herb in chronic fever. On long term use it is helpful in gaining weight in the emaciated persons and in losing weight in obese persons. It is reduces the ill effects of fat rich, creamy and oil food. It is used for curing swellings, skin and eye diseases. It can be used as home remedy against fever, cough, asthma and urinary disease. This herb has the ability to stop bleeding and prevent a medical condition called Hemorrhage. Its powder used as tooth paste, it will make your teeth stronger and healthy. Its paste with water is found to be anti-inflammatory, analgesic and having purifying and healing capacity of wounds [18]. Its decoction as a lotion is surgical dressing for healing the wound earlier. The fresh fruit taken before meals stimulates digestion, whereas if taken with meals it increases intelligence, nourishes the senses and purifies the digestive and genitourinary tract. Haritaki improves digestion, promotes the absorption of nutrients and regulates colon function. The plant also has adrenergic function and helps to recover from stress. The dried fruits of Terminalia chebula are used to produce the dye. The appearance of dye powder is brown and the main colouring component is chebulinic acid and its helps to remove toxins and unwanted fat from the body. Act as an effective anti-bacterial, anti-fungal, improves skin glow and complexion.

Medicinal uses of Terminalia chebula

It is good to increase the appetite, as digestive aid liver stimulant, as stomachic, as gastro intestinal prokinetic agent and mild laxative. It is stimulates the liver and protects it further by expelling the waste excretory products from the intestines. It is indicated in protracted diarrhea with hematochezia and prolapse of rectum. It is a good nervine, used in nervous weakness, nervous irritability. It promotes the receiving power of the five senses. It is helpful in renal calculi, dysurea, and retention of urine and used for treating parasitic infection. It is used as a blood purifier, gargle for sore throat, ulcerated gums, and muscular rheumatism. With sugar water it is used to treat opthalmia, skin itching and edema. It is used as an antioxidant, neuroprotective drug and treatment for heart disease, inflammation, brain dysfunction. It is used as an anti-aging agent and it is found to

improve the mental faculties. The plant also has adrenergic function and helps to recover from stress. One compound chebulagic acid from Haritaki has shown antispasmodic action like papaverine. The plant used is used as an antidote against bites of snakes. Regular use of Haritaki improves memory due to beneficial effects on the nerves of brain. The plant is used as a adjuvant in a herbal preparation called as “Triphala”. It is used as a Rasayana drug in Indian system of medicine (ISM). Triphala ^[26]is a Sanskrit name which means ‘three fruits’. The three plants used in making triphala are Terminalia chebula, Terminalia bellerica, Emblica officinalis. This is used in the proportion of 1:1:1 as per the Ayurvedic Formulations of India (AFI). This formulation is used as laxative in chronic constipation, detoxifying agent of the colon, food digestive problems (poor digestion and assimilation) and rejuvenator of the body. Terminalia chebula has been reported to protect the epithelial cells against influenza A virus infection and it also used to inhibit immediate hypersensitivity reaction that confirms its role in allergic conditions.

Effect on central nervous system

It is a good nervine. It is used in nervous weakness, nervous irritability. It promotes the receiving power of the five senses.

Effect on lungs

It is good for chronic cough, coryza, sore throat and asthma. It is used with other herbs in many holistic herbal formulations in ayurveda.

Effect on reproductive system

Being anti-inflammatory and astringent, it is useful in urethral discharges like spermatorrhea, vaginal discharges like leucorrhea. It can be given adjuvant in atonic conditiond of uterus.

Effect on kidney & urinary bladder

It is helpful in renal calculi, dysuria and retention of urine.

Effect on skin disrders

It is useful in skin disrders with discharges like allergies, urticarial and other erythematous disrders.

Pharmacological Activities

Anti-oxidant activity

Terminalia chebula is found to be an excellent anti-oxidant. The aqueous extract of T. chebula was tested for its potential antioxidant activity by means of its ability to inhibit gamma-radiation-induced lipid peroxidation in the tested rat liver microsomes and the damage to superoxide dismutase enzyme in rat liver mitochondria. On irradiation, the activity of the enzyme was found to be decreased which proves the restoring activity of the enzyme to a great extent. These studies confirm that the extract of T.chebula was able to protect the antioxidant enzyme from the effect of the reactive oxygen species that is produced by gamma radiation. The methanol, water and 95% ethanol extracts from Terminalia chebula were found to have the greater antioxidant activities. The higher antioxidant activities were observed in the methanol and 95% ethanol extracts of Terminalia chebula. The ethanolic extracts of the frtuits of Terminalia was also tested to know its anti-oxidant activity in albino rats and was found to decrease the levels of lipid peroxidase. T.chebula have the capacity to show the protection against the photosensitization-induced oxidative damage as they show their ability to prevent the process of Fe(II)-induced lipid peroxidation that might be inturn useful in reducing the photo-induced iron toxicity.

Anti-bacterial activity

Terminalia chebula is proven to be an effective anti-bacterial agent. Among the ether, alcohol and water extracts of T.chebula, ether extract was found to be very effective with Minimum Inhibitory Concentration and Minimum Bacteriocidal Concentration. Terminalia proves to be an effective anti-bacterial agent by forming the inhibitory zone against Pseudomonas aeruginosa, P. fluorescens, B. bronchiseptica, S. aureus, S.epidermidis, B. cereus and B.pumilis. Terminalia was found to be effective against both gram- positive and gram-negative bacteria and was confirmed to act as an excellent antimicrobial agent against the tested organisms such as Bacillus subtilis, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli K-12 and Staphylococcus aureus.

Anti-fungal activity

Terminalia chebula is expected to act against the fungal infection. A study was conducted on the anti-fungal activity of Terminalia sp. In that study aqeous, ethanolic and alcoholic twig extracts were tested against the fungal strains Alternaria brassicicola, A. alternata, Helminthosporium tetramera, Aspergillus flavus and A.niger. Results showed that aqeous extracts were not much effective. Alcoholic extracts showed better inhibition than aqeous and ethanol extracts. It is also found that A.niger was better inhibited by T.chebula. Another study was made on the inhibitory action of 42 methanolic plant extracts including the above plant over the Clotrimazole-resistant Candida albicans and Aspergillus flavus and was found that the methanolic extract of Terminalia chebula unripe seed inhibited the fungal infection.

Anti-cancerous activity

Terminalia is proved to possess anti-cancerous activity. The 70% methanolic extracts of the plant against five different cell- lines such as human prostate cancer cell line (PC-3), human (MCF-7) and mouse (S115) breast cancer cell lines human osteosarcoma (HOS-1) and a non-tumorigenic, an immortalized human prostate cell line (PNT1 A) was tested. The compounds responsible for cytotoxic activity such as chebulinic acid, ellagic acid and 2,4- chebulyl-b-D glucopyranose was also isolated. The highest activity was shown on PNT1A cell lines and PC3 cell lines. The compound chebulagic acid was evaluated for its capacity to inhibit the growth of the five cell lines such as MDA-MB-231(breast carcinoma), HCT-15, COLO-205 (colon cancer), DU-145 (prostatecancer) and K-562 (chronicmyeloidleukemia) and was found to be positive. Chemomodulatory effect of Terminalia chebula against the nickel chloride was tested with the methanolic extract on Wistar rats and was found out that the given extract down regulates the GSH, and GR activities. Another study was made on the ability of Triphala to inhibit Cytochrome P450. They found that the inhibitory activity against CYP3A4 had IC50 values of <0.1 mg/ml for ethanol. Mice grafted with human pancreatic tumours fed the Triphala formulation showed the reduction in the size of the tumours to half the size of those in a control group of mice that were fed with saline.

Anti-diabetic activity

Terminalia was found to have anti-diabetic activity. The extracts of Terminlia for its anti-hypoglycemic and anti-diabetic activity was conducted against the advanced glycation end products (AGEs)-induced endothelial cell dysfunction and was found out that the treatment of chebulic acid reduced glycer-AGE induced formation. The methanolic and ethanolic extracts of the plant was found to reduce the glucose levels.

Anti-inflammatory activity

Anti-inflammatory activity was expected to be present in T.chebula and was tested in the Swiss albino mice. Triphala treatment was found to significantly inhibit the paw volume and also the levels of lysosomal enzymes, lipid peroxidation and inflammatory mediator tumour necrosis factor. However the anti-oxidant status was found to show an increase in the plasma, liver andspleen of monosodium urate crystal-induced mice on comparision with the control mice. ß-glucuronidase and lactate dehydrogenase level were also found to be reduced in Triphala treated monosodium urate crystal-incubated polymorpho nuclear leucocytes. Results obtained from the above studies clearly indicated that Triphala possess a strong anti-inflammatory effect againstgouty arthritis.

Anti-viral activity

Terminalia was found to possess an excellent anti-viral activity against the cytomegalo virus. The hot water extract of the plant inhibited the Plaque formation of HCMV without depending on the dosage. Anti-HCMV or anti-MCMV activity was found out at the much lower concentration and was determined that Terminalia chebula significantly suppressed the CMV yields in the lungs of cyclosporine-treated mice on comparision with the water treatment. The study performed with T. chebula extracts on combination with the acyclovir against herpes simplex virus 1 showed their anti-HSV-1 activities very strongly. Terminalia was found to be the potentially inhibitor of swine influenza virus. The acetone extract of Terminalia chebula could be considered as the effective method for human beings who are fighting against pandemic swine influenza A virus due to its low cost, easy preparation and significant therapeutic potential.

Wound healing activity

The hydroalcoholic extract of T.chebula fruit was tested for its wound healing activity in the alloxan induced diabetic rats by using the excision and dead space wound modelswhich showed a significant increase in the wound healing activity in the fruit extract treated rats. The T.chebula extract was found top romote the healing of wound contraction in alloxan induced diabetic rats when applied topically by means of increasing the rate and extent of wound closure. The wound healing activity of the ethanolic extract of fruit of Terminalia chebula was also evaluated on excision and incision model in the albino rats in the form of an ointment.

Anti-ulcer activity

The anti-ulcer activity of the methanolic extract of Terminalia chebula fruits were evaluated in the pylorus ligation and ethanol induced ulcer models of the wistar rats proved them to be a potent anti-ulcer agent. Histopathologicalchanges observed on the pylorus ligation model have showed the degeneration, hemorrhage, edematous appearance of the gastric tissue.

Anti-caries activity

The aqueous extract of Terminalia chebula was investigated for its ability to inhibit the growth and physiological functions of Streptococcus mutans. The aqueous extract of T. chebula was found to possess the antibacterial activity against S. mutans with the Minimum Inhibitory Concentration. Mouthrinsingwith the solution of the extract of T. chebula brought resulted in a significant reduction in the total salivary bacterial count as well as the total streptococcal count. This proves that the extract of T. chebula can be used as an effective agent for the treatment of carious teeth because of its ability to inhibit the growth and accumulation of S. mutans on the tooth surface which would prevent the accumulation of acids, demineralization and the breakdown of the tooth enamel.

Radioprotective activity

The aqueous extract of triphala was administrered intraperitoneally to study the radiation-induced mortality in the mice that were exposed to g-radiation. The study found out the fact that the optimum radioprotective dose of 10 mg/kg the T. chebula was found to be 1/28 of the LD50 dose (280 mg/kg b.wt.), which was found to be far below the LD50 dose.

Cytotoxic activity

The cytotoxic effect of acetone extract of “Triphala” was tested on the cancer cell-lines using Shionogi 115 (S115) and MCF-7 breast cancer cells and PC-3 and DU-145 prostate cancer cells as the models. The cytotoxic assay was done on the 115, MCF-7, PC- 3 and DU-145 cells by means of measuring the [3H] thymidine labeling and was found to be positive against PC-3 cells.The chronic toxicity of the water extract from the dried fruits of T. chebula was orally administrated in rats in order to evaluate their safety and was concluded that they donot produce acute and chronic toxicities.

Antiarthritic Effect

The antiarthritic potential of Terminalia chebula has been evaluated in mouse model of arthritis. The suppression of the onset and progression of collagen induced arthritis by chebulagic acid obtained from the immature seeds of Terminalia chebula confirms its antiarthritic potential with a mechanism involving suppression of T cell activity. This study examined the effectiveness of T.chebula extract against the onset and progression of collagen-induced arthritis in mice.

Immunomodulatory Effect

Immunosuppressive effects of gallic acid and chebulagicacid, the active phytoconstituents of Terminalia chebula extract, on cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. It has been noted that gallic acid and chebulagic acid blocked the CTL- mediated cytotoxicity. Moreover, gallic acid and chebulagicacid has been shown to inhibit the killing activity of CD8+ CTL clone at IC50 values of 30 microM and 50 microM, respectively. Additionally, the granular exocytosis in response to anti-CD3 stimulation was also blocked by gallic acid and chebulagic acid that further evidenced its immunosuppressive effect.

Anti-typhoidal activity

The Aqueous extract was evaluated for its ability to reduce the risk of typhoid fever in Swiss albino mice. The extract was found to exhibit anti salmonellae activities against S.typhi and S. typhimurium by means of the clear zone of inhibition. Terminalia acts both as the bacteriostatic and as the bactericidal agent.

Cardioprotective activity

Terminalia has been proved to be an efficient cardioprotective agent. The alcoholic extract of T. chebula pretreatment in order to attenuate the isoproterenol induced alterations on the heart mitochondrial ultrastucture and function in the experimental rats was investigated. Terminalia chebula extract pretreatment provided significant protection against the metabolic alterations induced by the ISO (isoproterenol). Another study was conducted to evaluate the therapeutic efficacy of T. chebula in the protection against the isoproterenol induced lysosomal membrane damage which was found to be positive. The study confirmed that the pretreatment with T. chebula extract has partly impart its cardioprotective effect by means of the lysosomal membrane stabilization and thus prevents the myocardial necrosis.

LITERATURE REVIEW

In 2012

1. Mahdia Rahman et al., Reported that aqueous extract of Terminalia chebula, a natural medicinal herb had been demonstrated to exert its bactericidal activity against Salmonella sp., Shigella sp., Vibrio cholera and Escherichia coli: the bacteria that cause gastroenteritis. This activity was analyzed upon addition of different concentrations of salts of magnesium and manganese in the aqueous extract of the plant in order to observe the effect of the salts, if any. This study revealed that addition of manganese chloride at a concentration of about 80µg/gm of dried leaf powder could further increase the existing anti-bacterial activity of the plant to enteric bacteria by 25 to 45 %.

2. Pervaiz Ahmad Dar et al., Says that the present study was to evaluate anticatalep effect of Halelasiyah (Terminalia chebula) extract, on haloperidol induced catalepsy in swiss mice which were divided into four groups of six animals each. Animals in different groups were administered distilled water, scopolamine (1.0 mg/kg p.o) and hydroxy alcoholic (3:7) extract of Halelasiyah (Terminalia chebula), 1.5gm/kg and 3gm/kg p.o. respectively. Catalepsy was induced with haloperidol (1.0 mg/kg i.p.) administered 30 minutes prior to the drug administration. The duration of cataleptic time in the animals was assessed at 30 minute intervals up to 120 minutes and at the end of 240 minutes. Haloperidol induced catalepsy test was carried out for single dose study and after seven days of drug administration, to assess time and dose dependent effect of the test drug. The results showed that after the haloperidol administration, Halelasiyah (Terminalia chebula) extract in different doses was effective in reducing the cataleptic time significantly (p<0.01) in a time and dose dependent manner. The present study indicated that Bisfaij facilitates dopaminergic transmission and possibly acts as D2 receptor agonist and hence can be developed as an alternative/adjuvant drug in preventing and treating the extrapyramidal disorders.

3. R Rathinamoorthy et al., Reported as the study was focused on treatment of water and methanol extracts of Terminalia chebula fruits and citric acid as a cross linking agent on cotton plain-woven fabric. The active antimicrobial compounds in extracts were analyzed by high performance liquid chromatography. The fabric samples were tested for antibacterial activity against bacterial Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Salmonella typhi under agar diffusion strains like test and quantitative analysis. The results indicated that the treated cotton fabric shows a clear microbial resistance with 27-38mm zone of inhibition in the agar diffusion test against all the above mentioned strains. The treated samples showed 99% of reduction against Staphylococcus aureus and 86.25% reduction against Escherichia coli as per quantitative analysis. The Fourier Transform –Infra Red analysis confirmed the presence of active substances (saponin, ascorbic acid and gallic acid) in the treated samples. Process parameters were optimized using the response surface methodology adopted using Box Behnken design and the correlation coefficient was found to be 0.932 in the case of Staphylococcus aureus and 0.66 in the case of Escherichia coli.

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