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 Table of Contents  
Year : 2021  |  Volume : 6  |  Issue : 4  |  Page : 218-238

Antiproliferative potential of Amalaki Rasayana and the effect of Butea monosperma (Lam.) Taub on the cytotoxicity

1 Chemical Sciences and Technology Division, CSIR—National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
2 Chemical Sciences and Technology Division, CSIR—National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India
3 Department of Rasasastra and Bhaishajya Kalpana, Government Ayurveda College, Thiruvananthapuram, Kerala, India

Date of Submission05-Nov-2021
Date of Acceptance29-Jan-2022
Date of Web Publication17-May-2022

Correspondence Address:
Kokkuvayil V Radhakrishnan
Chemical Sciences and Technology Division, CSIR—NIIST, Thiruvananthapuram 695019, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jdras.jdras_71_21

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BACKGROUND: Amalaki Rasayana is one of the prominent rejuvenating Rasayana described in Indian traditional Ayurvedic medicine for healthy aging. AIM: This work is focused on the comparative evaluation of the antiproliferative potential of AR, amla (a constituent), and Butea monosperma (BM; a component in the preparation) in the human cervix adenocarcinoma (HeLa) cell line and normal lung fibroblast (WI-38) cell lines. Also, we carried out the identification of phytoconstituents from the heartwood of BM. MATERIALS AND METHODS: Cell growth inhibitory effects of the extracts of AR, amla, and BM were carried out using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Isolation and characterization of compounds from BM were carried out using column chromatography and various spectroscopic techniques. RESULTS: AR exhibited a significant anticancer activity in HeLa cells compared to amla, and the Rasayana was found to be less cytotoxic toward normal cells. The results indicated an increase in the cytotoxicity to HeLa cells when amla is processed compared to AR in the heartwood of BM. The phytochemical investigation of BM revealed the presence of isoflavones as the major constituent. The isolated compounds were formononetin, daidzein, prunetin, lupiwighteone, afrormosin, erypoegin K, genistein, sterols β-sitosterol and stigmasterol and a monosaccharide d-mannitol. Erypoegin K, lupiwighteone, and d-mannitol were reported for the first time from this species and afrormosin was reported for the first time from the heartwood. CONCLUSIONS: Antiproliferative potential of AR was confirmed on cervical carcinoma. BM significantly enhances the therapeutic potential of AR, and AR could be an effective chemopreventive agent.

Keywords: Amalaki Rasayana, Amla, Butea monosperma, cytotoxicity, isoflavones

How to cite this article:
Subramanyan S, Selvakumar D, Omanakuttan VK, Maiti KK, Varma RL, Pillai RV, Radhakrishnan KV. Antiproliferative potential of Amalaki Rasayana and the effect of Butea monosperma (Lam.) Taub on the cytotoxicity. J Drug Res Ayurvedic Sci 2021;6:218-38

How to cite this URL:
Subramanyan S, Selvakumar D, Omanakuttan VK, Maiti KK, Varma RL, Pillai RV, Radhakrishnan KV. Antiproliferative potential of Amalaki Rasayana and the effect of Butea monosperma (Lam.) Taub on the cytotoxicity. J Drug Res Ayurvedic Sci [serial online] 2021 [cited 2022 Jul 7];6:218-38. Available from: http://www.jdrasccras.com/text.asp?2021/6/4/218/345398

  Introduction Top

Ayurveda, the traditional Indian system of medicine, signifies the science of long life. It revolves around three basic senses of humor of the body, i.e., Vata (body movement), Pitta (transformation), and Kapha (support and growth) called the tridoshas. These three factors in equilibrium are the state of health, and an imbalance in this stability causes diseases.[1] The treatment of Ayurveda targets to bring the vitiated dosha (regulatory functional factors of body) back to the state of equilibrium. Sushruta defines a healthy man as one who has an equilibrium of doshas, normal condition of seven Dhatus (major structural components of the body), normal functioning of Agni (digestive/metabolic factors), and besides his soul, the sense organs and mind should be happy and cheerful.[2] Rasayana (rejuvenative) therapy is an active step toward achieving this, and it constitutes one of the eight major divisions of Ayurveda. Rasayana includes a set of specific therapies that can reverse the physical degeneration of the body caused by aging.[3]Rasayana chikitsa (“Rasa”: plasma; Ayana: path, which means the path that “Rasa” takes) or therapy improves longevity, intelligence, memory, health, youth, voice, complexion, motor, and sensory strength.[4] According to sages, rejuvenation therapy is of two types (i) Kutipraveshika and (ii) Vatatapika. The first one is the most beneficial treatment. “Kuti” is a specially built hut in which no light enters and the patient is sequestered there for a long time by taking the various Rasayana herbs.[5]Amalaki Rasayana (AR) is one of such rejuvenation medicines prepared from amla (Emblica officinalis Gaertn. [EO] or Indian gooseberry). The Rasayana formulation mentioned in Charaka Samhita was prepared by heating the raw fruits of amla in a covered tube made of fresh Palash wood (Butea monosperma [BM]), pasted with mud and cooked with wild cow-dung fire. After self-cooling, the fruits were taken out, and seeds were removed and crushed. They are then mixed with pippali powder, vidanga powder, sugar, oil, honey, and ghee. The preparation is stored for 21 days thereafter for the Rasayana to mature before it is used.[6]

AR was used since time immemorial by sages or ascetics to extend their lives in order to achieve liberation in their lifetime. AR is grouped under Vayasthapana Rasayana (medicine which prolong), which promotes longevity and healthy aging. Previous studies on AR indicated that it possesses significant immunostimulant activity and moderate cytoprotective activity.[7] AR enhances the telomerase activity in aged individuals, delaying the onset of the aging process.[8] The combination of AR and milk is very effective in treating aging elements.[9] AR alleviates suffering from neurodegenerative disorder like Alzheimer’s disease and promotes healthy aging with enhanced physical and mental strength.[10] Dietary supplements of AR and Ras Sindoor in Drosophila models substantially suppressed the neurodegeneration in fly models of polyQ and Alzheimer’s disorders without any harmful side effects.[11] Apoptotic death of the affected neurons is one of the prominent features of neurodegeneration. These formulations effectively suppressed the apoptosis induced and elevated the antiapoptotic proteins’ levels.[12] AR feeding in flies Drosophila melanogaster significantly improved the oxidative stress tolerance and reduced the accumulation of reactive oxygen species (ROS) with age, reflecting improved cellular redox homeostasis.[13] AR triggers a protective effect on ultraviolet-induced DNA damage in aged individuals without altering the nucleotide excision repair and constitutive base excision repair.[14] It is also efficient in enhancing cardiac function and the management of anemia (Pandu).[15],[16]

As mentioned in the literature, AR is prepared in a wooden vessel made of Palash (BM). Palash sticks and wood were used in different Indian rituals from ancient times. The plant is used in Ayurveda, Siddha, Unani, and Homeopathy medicine for various ailments and has become a treasure of modern medicine. Palash, popularly known as the “Flame of the forest,” belongs to the family Fabaceae. BM is a native plant of the Indian subcontinent and southeast Asia. It is said that the tree is a form of Agnidev “The God of Fire” and is believed to be a sacred tree. BM has been traditionally reported to possess antibacterial, antimicrobial, antifungal, astringent, anticonceptive, anticonvulsant, antitumor, antifertility, hepatoprotective, radical scavenging, and wound-healing activity.[17]

Chemoprevention with food phytochemicals is considered as one of the most important strategies to control cancer. EO possesses potent free radical scavenging activity, thereby preventing carcinogenesis.[18] As the preparation of AR uses the plants amla (EO) and Palash (BM), our study mainly aims at the comparative evaluation of the anticancer property of amla and AR along with the phytochemical reinvestigation of BM. An attempt has been made to validate the method of drug processing (heating amla inside Palash wood) and its influence on the therapeutic property of amla after the processing.

  Materials and Methods Top

General experimental procedure

Different analytical techniques were used for the characterization of the compounds. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AMX 500 spectrometer. Mass spectra were measured using Thermo Scientific Exactive mass spectrometer under electrospray ionization-high resolution mass spectrometry (ESI-HRMS) at 60,000 resolutions. Silica gel (230–400 mesh and 100–200 mesh) and Sephadex LH-20 were used for gravity column chromatography (CC). Silica gel 60 F254 aluminum-backed thin-layer chromatography (TLC) plates from Merck were used for analytical TLC. All the chemicals and solvents used were purchased from Sigma-Aldrich and Merck.

Plant materials

BM and EO were collected from the Paravur region of Kollam district of Kerala, India. Voucher samples of the plants were deposited at the Department of R and B, Government Ayurveda College, Kannur (voucher number EO: 2018-06-03 and BM: 2018-06-05).

Amalaki Rasayana preparation

As described in our previous work (Supporting Information).[19]

Extraction method for Rasayana

Fresh fruits of amla (EO) after removing seeds, powdered AR, and the stem bark of Palash (BM) were taken about 10 g each and extracted with 70% ethanol–water mixture at room temperature. Ethanol was removed under vacuum in a rotatory evaporator, and the remaining mixture was lyophilized to get the crude extract. The extraction yielded 484.6 mg of AR, 485 mg of EO, and 70.2 mg of BM extract. The extracts were analyzed for cytotoxicity in HeLa and WI-38 cell lines.

Extraction and isolation method for heartwood of Butea monosperma

The air-dried, coarsely powdered heartwood of BM (1.5 kg) was extracted three times with acetone (5 L × 3 days) at room temperature. The solvent was filtered and then concentrated under vacuum, which yielded 33 g of the crude extract. Further, the acetone extract was fractionated using silica gel (100–200 mesh) CC with gradient mixtures of hexane and ethyl acetate of increasing polarity. According to the similarity in the TLC spots, fractions were combined to give nine fraction pools (Fr.1– Fr.9). The fraction pools on further purification resulted in the isolation of compounds 110. Fr.2 on silica gel CC separation yielded two compounds (colorless needle-like crystals) labeled as compound 1 (10.8 mg) and 2 (7 mg). Fr.4 contains a white amorphous solid designated as compound 3 (30 mg). The residue after separating compound 3 subjected to silica gel CC using 30% ethyl acetate–hexane polarity afforded three compounds counted as compound 4 (5 mg), 5 (4 mg), and 6 (4 mg). Compound 7 was isolated from Fr.5 as a white solid (6.5 mg) on CC separation. Fr.6 showed a fluorescent nature that yielded a yellow crystalline solid labeled as compound 8 (5 mg). Fr.7 on silica gel CC separation yielded a yellow solid with some minor impurities, which was further purified by Sephadex LH-20 column, and the compound was designated as 9 (2 mg). Fr.9 on silica gel CC separation on 90% ethyl acetate-hexane polarity afforded an amorphous colorless solid, counted as compound 10 (22 mg).

Cytotoxicity analysis

As described in our previous work (Supporting Information).[20]

  Results Top


3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was performed to assess the cytotoxicity of all the extracts. We evaluated the extracts against human cervix adenocarcinoma (HeLa) and normal lung fibroblast (WI-38). Doxorubicin (DOX) was used as the standard drug. The results of the cytotoxic studies are shown in [Table 1]. The effects of various concentrations of EO, AR, and BM on HeLa and WI-38 cell lines at 24 and 48 h are shown in [Figure 1]. The results showed that AR and BM possessed potent cytotoxic activity against HeLa cells with a half-maximal inhibitory concentration (IC50) value falling 2.94 and 0.545 μg/mL, respectively, at 48 h. After 48 h of incubation, 10 μg/mL of EO, AR, and BM extracts showed 26.85%, 65.70%, and 69.75% of inhibition on cell proliferation, respectively. The IC50 values for 24 h treatment were 13.8, 65.23, 44.77, and 1.56 μg/mL, respectively, for DOX and extracts. In the case of WI-38 cells, all the extracts showed the least cytotoxic effect than DOX. The IC50 values of the extracts were >100 μg/mL at 24 h and those of AR remained the same at 48 h of incubation. However, EO and BM exhibited cytotoxicity with an IC50 value of 33.43 and 22.72 μg/mL, repectively.
Table 1: Cytotoxicity data for different extracts in HeLa and WI-38 cells over a time of 24 and 48 h

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Figure 1: Evaluation of cytotoxicity of extracts by MTT assay on (A) HeLa cells at 24 h, (B) HeLa cells at 48 h, (C) WI-38 cells at 24 h, and (D) WI-38 cells at 48 h

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Phytochemical investigation of the heartwood of Butea monosperma

Phytochemical investigation of the acetone extract of the medicinally important plant BM was carried out. We have isolated 10 compounds [Figure 2] from the acetone extract of the heartwood of BM and successfully characterized all the molecules by extensive spectroscopic analysis. On the basis of NMR spectral data, HRMS analysis (see Supporting Information) and on comparison with literature reports, the structure of the compounds were elucidated as β-sitosterol (1), stigmasterol (2),[21 formononetin (3)],[[22] daidzein (4),[23] prunetin (5),[24] lupiwighteone (6),[25] afrormosin (7),[26] erypoegin K (8),[27] genistein (9),[28] and d-mannitol (10).[29] Erypoegin K, lupiwighteone, and d-mannitol were reported for the first time from the genus Butea. Afrormosin was isolated for the first time from the heartwood of BM.
Figure 2: Chemical structure of the compounds isolated from the heartwood of BM

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  Discussion Top

Amalaki or amla (EO) is the Indian gooseberry that belongs to the family Euphorbiaceae; it has traditionally been used for various medicinal purposes. According to the two primary classic texts on Ayurveda, Charaka Samhita, and Sushruta Samhita, amla is regarded as the best among rejuvenating herbs and the best among the sour fruits, and is useful in relieving cough and skin diseases.[30] This plant has an inevitable role in Ayurveda, having antioxidant, antitumor, hepatoprotective, diabetic, cardioprotective, anti-ulcer, oxidative stress, and memory-enhancing ability.[31],[32] The phytochemicals from the fruits include phyllemblin, phyllemblic acid, gallic acid, emblicol, quercetin, hydroxymethylfurfural, ellagic acid, pectin, and putranjivan A.[33]

Amalaki is referred to as the Maharasayana because the fruits serve as the major ingredient or one of the ingredients in most of the Rasayana preparation.[34] The classical Ayurvedic drugs such as Amritaprasham, Brahma Rasayana, Ashwagandha Rasayana, Narasimha Rasayana, Chyavanaprasha, and Triphala were reported to exhibit radioprotective effects.[35] All the Rasayana’s have a unique formulation with a number of plants and herbs, and the Rasayana treatment has multidimensional benefits other than memory enhancing, antiaging, etc. Vyas et al. reported the efficacy of the Rasayana Avaleha as an adjuvant medication to modern radiotherapy and chemotherapy. The preparation constitutes the plant species Amalaki, Ashwagandha (Withania somnifera (L.) Dunal), Yashtimadhu (Glycyrrhiza glabra), Guduchi (Tinospora sinensis (Lour.) Merr.), Tulasi (Ocimum sanctum L.), Jivanti (Leptadenia reticulata (Retz.) Wight & Arn.), and Pippali (Piper longum L.).[36]BrahmaRasayana is reported to possess immunomodulatory, anti-stress, adaptogenic, and memory-enhancing effects. It constitutes more than 35 ingredients in which Amalaki and Haritaki (Terminalia chebula Retz.) serve as the major ones.[37]Triphala consists of the dried fruits of Haritaki, Bibhitaki (Terminalia bellirica (Gaertn.) Roxb), and Amalaki in equal proportions. Triphala inhibits cancer cell proliferation in colon cancer, breast cancer, pancreatic cancer, etc.[38],[39]

The present study evaluates the potential of AR, EO, and BM for cytotoxicity against cancer cells and normal cells in comparison with the standard drug DOX. The extracts were assessed for their in vitro cytotoxicity against the human cervix adenocarcinoma (HeLa) cell line and showed moderate to excellent cytotoxicity as reflected in the IC50 value, i.e., 50.77, 2.94, and 0.545 μg/mL, compared to the control DOX (IC50 1.21 μg/mL). Moreover, all the extracts showed lower toxicity toward the normal lung fibroblast (WI-38) cells. The studies revealed the cell-specific cytotoxic effect of the extracts. The results found that AR and BM exhibited an appreciable cytotoxic effect toward HeLa cells even at a concentration of 2.94 and 0.545 μg/mL at 48 h while showing much less cytotoxic effect in WI-38 cells. AR showed much better cytotoxicity (2.94 μg/mL) than EO (50.77 μg/mL) at 48 h toward the HeLa cell line. In the case of WI-38 cells, AR was nontoxic up to a concentration of >100 μg/mL, whereas EO exhibited slight toxicity (33.43 μg/mL) at 48 h. The variation in cytotoxicity indicated that the processed amla (AR) showed more anticancer potential than raw amla.

The cytotoxicity analysis showed that BM was exhibiting excellent cytotoxicity than AR and EO toward cancer cells. To check whether the plant BM has any influence on the therapeutic property of amla, we need further studies. Hence, we carried out the phytochemical investigation of the heartwood of BM, which resulted in the isolation of 10 compounds. The compounds include seven isoflavones (formononetin, daidzein, prunetin, lupiwighteone, afrormosin, erypoegin K, and genistein), a monosaccharide d-mannitol and two sterols β-sitosterol and stigmasterol. The isoflavones isolated were reported for potent anticancer activity. Formononetin exhibits excellent cytotoxicity toward various cancer cells such as breast cancer, lung cancer, bladder cancer, prostate cancer, ovarian cancer, and cervical cancer.[40],[41] Afrormosin was previously reported from the flowers of BM,[42] heartwood of Gliricidia sepium (Jacq.) Walp.,[43] stem bark of Dipteryx alata Vogel,[26] and stems of Millettia dorwardi Collett & Hemsl.[44] of the Leguminosae family. This is the first report from the heartwood of BM. Afrormosin has antioxidant, anti-inflammatory, and antiproliferative activities.[45],[46] This is the first report of erypoegin K, lupiwighteone, and d-mannitol from the genus Butea. Erypoegin K was first reported from the stem bark of Erythrina poeppigiana (Walp.) O.F.Cook (Fabaceae) and possessed a significant apoptosis-inducing effect against human leukemia HL-60.[47] Lupiwighteone previously isolated from Vatairea guianensis Aubl. (Fabaceae) and Glycyrrhiza glabra L. has been reported to induce apoptosis in breast cancer cells MCF-7 and MDA-MB-231.[25],[48] Lupiwighteone inhibits cell growth and induces apoptosis in human neuroblastoma and prostate cancer cells.[49],[50] Genistein and daidzein, known as phytoestrogens, have been explored as growth inhibitors of several cancer cell lines.[51],[52] Both induce antiproliferative effects in prostate and breast cancer cells via the inhibition of different signaling pathways.[53],[54]

From the literature review, we could summarize that isoflavones play a significant role in contributing to the anticancer property of the species BM. BM showed high cytotoxicity than AR toward the cancer cell lines but exhibited some toxicity in normal cells. There is significant variation in cytotoxicity of amla and AR. The exact mechanism is not known; however, our results suggest that BM significantly contributes to the drastic change in cytotoxicity of AR. Moreover, AR has proven to be nontoxic to the normal cells, whereas amla showed toxicity. Thus, the method of such drug processing helps to reduce the toxicity of the raw fruits of amla. These findings suggest that the cytotoxicity of BM synergetically enhances the antiproliferative activity of AR, or in other words, BM helps to boost the therapeutic potential of amla. Also, the nontoxic nature of AR gives an immense advantage that AR could be an effective drug lead in chemoprevention along with other Rasayanas. Hence, the rejuvenation therapy with AR will help to improve the quality of life of cancer patients.

  Limitations of the Study Top

The present in vitro study showed the anticancer property of AR in Hela and WI-38 cell lines. Further experiments are necessary to elucidate the mechanism of action of AR in cancer cells. Also, in vivo experiments should be conducted for further scientific validation of the Rasayana and ingredients.

  Conclusions Top

The present work was undertaken to assess the potential of AR as a chemopreventive agent. AR and BM exhibited significant cytotoxicity toward the human cervix adenocarcinoma (HeLa) cell lines. Even though AR is not as effective as DOX against HeLa cell lines but exhibited low toxicity against normal lung fibroblast (WI-38) up to a concentration of 100 μM. Similarly, extracts of BM and EO showed lower toxicity in WI- 38 cells. The results indicated that the cytotoxicity of amla toward normal cells is reduced when it is thermally processed inside the wooden vessels of BM stem. So BM plays the role of a perfect synergistic therapeutic partner for amla to improve its therapeutic efficacy and reduce toxicity. It is evident from our study that the methods used by our ancestors for drug processing help to increase the therapeutic potential of the final drug form. Moreover, further studies are needed to uncover the exact mechanism of action of AR.


Thanks are du e to University Grants Commission (UGC)—Rajiv Gandhi National Fellowship (RGNF), New Delhi, acknowledged for research fellowship. We thank Mrs. S. Viji and Mrs. Saumini Mathew of CSIR-NIIST for the technical support in recording the mass and NMR spectral analysis.

Financial support and sponsorship

This study was financially supported by UGC—RGNF, New Delhi.

Conflicts of interest

The authors report no declarations of interest.

Data availability statement

The data set used in the current study is available on request from the corresponding author.

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  [Table 1]


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