Rasa: Madhura, Amla, Katu, Tikta, Kashaya

Guna: Laghu, Ruksha

Virya: Ushna

Vipaka: Madhura [Dravyaguna Vijyana, Vol.- II page-753]

Karma: It acts as Tridosha Shamaka (mitigates Vata, Pitta and Kapha). Shothahara (anti- inflammatory), Chakshushya (beneficial for eyes), Mukharogahara (cures oral diseases), Balya (strengthening), Deepana (appetizer), Paachana (digestant), Vatanulomana (carminative), Vruna Ropana (wound healing), Yakrita-Pleeha Uttejaka (hepatoprotective), Kasa -Swasahara (cures cough and breathlessness), Hikka (cures hiccough), Mootrala (act as diuretic), Rasayana (rejuvenator), Vajikara (aphrodisiac). [Dravyaguna Vijyana, Vol.- II page-753]

Therapeutic Uses - Shotha, Arsha, Aruchi, Hrydroga, Kasa, Pandu, Prameha, Udhvarta, Vibandha, Vishamajvara, Shiroroga, Tamaka Swasa, Gulma, Udararoga. [Dravyaguna Vijyana, Vol.- II page-753]

Useful part: Fruit, Root and Bark

Important Formulations -Triphala Choorna, TriphaladiTaila, Abhayarishta, Agastya Haritaki Rasayana, Chitraka Haritaki, Danti Haritaki, Dashmul Haritaki, Bramha Rasayana, Abhaya Lavan, Pathyadi Lepa. [Dravyaguna Vijyana, Vol.- II page-753]

Dose– Fruit Powder – 2-6 gm Bark Powder – 1-4 gm Kwath - 25 – 50 gm. Arishta – 2 ½ Tola (30ml) [Raj Nighantu]

Traditional values of Haritaki – In Charaka Samhita and Sushruta Samhita, various medicinal plants has been mentioned but Haritaki is placed in prime among medicinal plants. It is a top listed plant in Ayurvedic Materia medica for treatment of asthma, bleeding piles, sore throat, vomiting and gout (Aneja KR et al., 2009). It is used in Thai traditional medicine as a carminative, astringent and expectorant (Panunto W et al., 2011). According to Vagbhata, it is the drug of choice in the therapy of ‘Vata-Kapha’ diseases. The ‘Triphala’, a herbal formulation of ‘three fruits’ from plants Terminalia chebula, Terminalia Bellerica, Emblica officinalis, are used as laxative in chronic constipation, food digestive problems (poor digestion and assimilation) detoxifying agent of the colon, and rejuvenator of the body (Prasad L et al., 2006). The fruits of Haritaki are used both externally as well as internally for medicinal purposes. Externally, the paste of fruits effectively reduces the swelling, hastens the healing and cleanses the wounds and ulcers. In erysipelas and other skin disorders, Haritaki prevents accumulation of pus in skin diseases. The oil of Haritaki is extremely helpful in healing of wounds especially in burns. The paste of fruit is also applied in conjunctivitis for relief due to its anti-inflammatory property. The gargles with its decoction give excellent results in stomatitis and problems of the throat. A fine powder of Haritaki is used as a tooth powder to strengthen the gums. Internally, Haritaki is used to cure a vast of disease. According to Vagabhatta, when Haritaki powder fried in ghee is regularly consumed with sufficient ghee in food, it promotes longevity and boosts energy. Common gastrointestinal ailments, tumours, ascites, piles, enlargement of liver and spleen, worms, colitis can be treated well with Haritaki. ‘Bala Haritaki’ is useful in haemorrhoids and in clearing the bowels. In abdominal pain due to flatulence, it is given with jiggery and ghee. The most popular combination of Haritaki, Musta, Shunthi and jaggery is an effective panacea for diarrhoea, dysentery, and flatulence etc. ‘Haritaki Siddha Ghrita’ is beneficial in chronic fever. Haritaki powder with honey and ghee is also effective remedy for anaemia. In obesity, its decoction with honey reduces the excessive body fats. Regular use of Haritaki improves memory due to beneficial effects on the nerves of brain. It is also valuable in dysuria and urinary stones. (Kirtikar KR et al., 1935)

Fruits of Haritaki (Terminalia chebula Retz.) were procured from Organic Farm in Umbari west of Satara District in Mahararshtra.

Authentication and voucher deposition – It was done in the Botany Department of authorised Institute. A voucher specimen of the collected sample was deposited in the departmental museum for future reference.

Accession No. – HC0024

1. Material – Powdered form of fruit of Terminalia chebula. Choorna was prepared according to the guideline mentioned in Sharangdhar Samhita and subjected to standardization.

First of all CO₂ which was stored in tank is pumped to the extractor (Fig. 2) where pressure was maintained at 300 bar (=296.077atm) and at the temperature 304 K (31°C). In the extractor, liquid CO₂ and feed (Fig. 1) come in contact with each other and extraction of essential component from feed was carried out. The product steam which contains CO₂ and essential component goes to the cyclone separator through PRV (Pressure relieving valve), due to deduction in pressure PRV liquid CO₂ is converted into gas. In cyclone separator CO₂ gas and essential component (liquid form) are separated from each other. From the top of the cyclone separator CO₂ is recycled back to the tank, before sending it to the tank, CO₂ which turned to gaseous state is condensed with the help of condenser. From the bottom of the cyclone separator essential components i.e. CO₂ extract of T. chebula (Fig. 3) was obtained.

Cell line – MDA-MB-231 Breast Cancer Cell Line

Source – APT Research Foundation, Pune (Maharashtra)

Procedure -Hands was sprayed with 70% ethanol. Removed T-flasks from the incubator which contains cell lines. Checked cells in flask under microscope to confirm that the cells are 90% confluent. Old media/ spent media from flask was removed. Phosphate buffer saline (PBS) for eg.2 ml in T25 flask likewise 4ml/6ml PBS was added in T75/T225 flask respectively and shook thoroughly to detach all the cells. Within few second / immediately remove PBS or Pipette out from flask. Then trypsin was added for e.g. 1 ml in T25 flask likewise 2ml/4ml in flasks T75/T225 respectively. Then flask containing trypsin and cell lines were kept in incubator for 3 minutes (if kept more than 3 min cells will get stressed). After 3 min took the flasks from incubator and made sure that all the cells were suspended in the trypsin by gently tapping. Then added 5 ml of complete media in T25 flasks. For e.g. 15ml in T75 and 25 ml in T225 flasks. Then pipettes out in centrifuge tube or falcon tube. This was also simultaneously taken for cell counting and the result was noted. Centrifuge for 2-3 minutes the pellet is formed then took pellet and discard supernatant. Fresh media was added to the falcon tube and was pipettes in and pipettes out several times dislotch. For e.g. 5ml in T25 flask. For equal distribution of cells the flask was moved in infinity symbol pattern (). It was sealed by parafilm to prevent contamination. Labelled the flask by cell line, passaging number. The flask was kept in the incubator for 24 hrs. (Fig. 4)

CO₂ extract of Terminalia chebula was screened for the presence of various Phytoconstituents using standard procedures. In CO₂ extract of fruit of Terminalia chebula following constituent like Tannin, Alkaloid, Phenol, Carbohydrate, Steroids, Protein, Resin were detected.

HPTLC fingerprint of CO₂ extract of fruit of Terminalia chebula is little bit similar as of phytochemical analysis. The results indicate that Terminalia chebula contains a number of markers that may be responsible for its anticancer activity. The developed HPTLC method will assist in the standardization of Terminalia chebula using biologically active chemical markers. The proposed Validated HPTLC method for Gallic acid from Terminalia chebula seems to be accurate, precise, reproducible and repeatable. Terminalia chebula also contained a number of other constitute, which was shown in phytochemical analysis. Hence, the present study has been helped to given out the parameters to determine the anticancer activity of CO₂ extract of fruit of Terminalia chebula. When HPTLC study of CO₂ extract of Terminalia chebula was carried out with the help of Mobile phase Toluene: Ethyl acetate: Formic acid (3:6:1v/v) for Gallic acid. Prominent light bands detected at R_f value is 0.41 under at 280nm. The method was found to be very specific. Since the overlay spectrums of standard Gallic acid with sample of Terminalia chebula were found to be similar. Quantification of Markers present in Terminalia chebula was 0.9689% w/w.

Day 1 - 104 cells/well was added in a 96 well tissue culture treated plate (Cell count was taken on a Neubauer’s chamber). The plate was incubated at 37°C in a 5% CO₂ incubator for 24 hours.

Day 2 - After 24 hrs incubation, the plate was observed under inverted microscope to check the morphology of the cells and confluency of the wells in the 96 well plate. Sterile test sample was suspended in DMEM containing 10% FBS at a known concentration and dilutions for the same were made accordingly. 100 µl of each of the test samples were added in triplicates along with the positive control (DMSO) and normal control (cells with medium and no test sample). Post sample addition, the plate was incubated at 37°C in a 5% CO₂ incubator for 24 hours.

Day 3 - After 24 hours incubation, the plate was observed under the inverted microscope and photographs were taken of the recorded observations. Test sample was removed and 90 µl fresh DMEM containing 10% FBS was added. Then 10µl of MTT reagent was added in each well. The plate was wrapped in aluminium foil and incubated at 37°C in a 5% CO₂ incubator for 4 hours. Post 4 hours incubation, the entire medium was removed by flicking the plate and 100 µl of solubilisation buffer was added in each well and incubated at 37°C in a 5% CO₂ incubator for about 20 minutes. Post incubation, absorbance was measured at 630 nm on 96 well Plate reader.

The MTT assay was used for in vitro experiment to determine the effect of Terminalia chebula Retz. Extract on breast cancer cell line MDA-MB-231.has been carried out at the department of Ilmul Advia in Regional Research Institute of Unani Medicine (RRIUM), and University of Kashmir-Srinagar. The aerial parts of the plants were studied for their pharmacognostic standardization and anxiolytic activity.

ANOVA test was applied to prove the treatment statistically significant or not, as there were 7 groups for anticancer activity CO₂ extract of Terminalia chebula Retz. in MDA-MB-231 breast cancer cell line. (Table no.3 and Table no.4)

The mean difference is significant at the 0.05 level. It indicates that CO₂ extract of Terminalia chebula able to show positive results on MDA-MB-231 Cell line as compare to Standard drug DMSO. Hence, from the above discussion it can be stated that CO₂ extract of Terminalia chebula shows anticancer activity.