In Vitro Anti-cancer, Anti-hypertensive and Anti- hyperglycaemic effect of Hypoxis colchicifolia

Economic challenges associated with non-communicable diseases and the sociocultural outlook of many patients especially in Africa has increased the dependence on traditional herbal medicines for these diseases. Hypoxis colchicifolia is a traditional medicinal plant used in Southern Africa against an array of ailments. This study evaluated the in vitro antidiabetic (α-amyalse and α-glucosidase), antihypertensive (angiotensin-converting enzyme) and anticancer potential of H. colchicifolia corm as well as leaf (acetone, methanol and aqueous) extracts. Results showed that extracts have a moderate anti-diabetic and anti-hypertensive potential, with great anti-cancer potential. The acetone extract of both fresh and dried corms produced significant α-amylase and α-glucosidase inhibition with ACE inhibited predominantly by the dried corms methanolic extract (IC50 368.2 μg/mL). Methanolic extract of dried leaves showed the least cytotoxicity against the noncancerous cell line HEK-293 while exhibiting the highest inhibition of MCF-7 cells (IC50 3.24 μg/mL). All extracts exhibited a greater inhibitory potential in A549 cells than the positive control camptothecin (IC50 304.2μg/mL). This study reveals that H. colchicifolia has therapeutic potential as an anti-diabetic and anticancer agent; however, further in vivo studies need to be conducted. *Author for correspondence JOURNAL OF NATURAL REMEDIES DOI: 10.18311/jnr/2021/25315 Article Received on: 11.05.2020 Accepted on: 11.05.2021 RESEARCH ARTICLE Revised on: 14.12.2020


Introduction
Non-Communicable Diseases (NCD) are the leading cause of death globally, with the top killers that together accounts for more than 80% of all precipitate NCD deaths including hypertension (17.9 million deaths annually), cancer (9.0 million) and diabetes (1.6 million) 1 . Similarly, in South Africa, diabetes, cancer and hypertension remain the greatest cause of morbidity. Conventional treatment for each of these NCDs do exist, however, these drugs have numerous side effects. Furthermore, despite the prevalence and burden of these disorders, a large proportion of people with such problems do not receive treatment 2 . Treatment remain largely inaccessible predominantly in the developing countries due to their exuberant price tag as well as weaknesses in the health care systems, hence, they depend, even if nominally, on alternative therapies such as traditional herbal medicines.
In certain parts of Africa, traditional medicine remains the most employed method of healthcare

In Vitro Anti-cancer, Anti-hypertensive and Antihyperglycaemic effect of Hypoxis colchicifolia
Journal of Natural Remedies | ISSN: 2320-3358 http://www.informaticsjournals.com/index.php/jnr | Vol 21 (3) | July 2021 because of their accessibility to the community 3 . The importance of phytomedicines has been recently sparked scientific investigations, as the therapeutic functionality of medicinal plants is limited. Common plants that are effective and tested against hyperglycaemia are Panax ginseng (Ginseng), Momordica charantia (Bitter melon), Coptis chinensis, Trigonella foenum-graecum (Fenugreek), Lagerstroemia speciosa, Gymnemasylvestre, Cinnamomum cassia (Cinnamon) and Agaricus campestris mushrooms 4 . Kamtekar et al.,5 found that plant extracts that are rich in phytochemical secondary metabolites such as flavonoids and phenolics have the potential to control diabetes due to the alpha amylase inhibition potential of these compounds. Odhav et al., 6 found that traditional African vegetables such as Centella asiatica, Ceratotheca triloba, Cleome monophylla and others were effective in inhibition of α-amylase. Plant derived compounds such as terpenoids and polyphenolic are known to possess in vitro ACE inhibitory activities 7 . Ranilla et al., 8 found that peppers and spices (Cuminum cyminum, Zingiber officinale, Curcuma longa and Cinnamomum zeylanicum) had significant ACE inhibition due to the phenolic compounds present and can significantly aid in lowering hypertension. About 60% of anticancer agents that are currently used come from natural sources 9 . These include vinca alkaloids, taxanes, podophyllotoxin, campothecin, anthracyclines 10,11 . However, the quest to find the ideal anticancer drug, which kills cancer cells while having minimal effect on normal cells, endures. Hypoxis colchicifolia is commonly referred to as broad leaved Hypoxis, 'inkomfe' , 'igudu' , 'ingcobo' and 'ilabatheka' , in Zulu. It is one of the four most sought after plant species in traditional medicine. H. colchicifolia corms are used against barrenness, heart weakness and bad dreams. Infusions of the corm are drunk in small quantities as a tea to stop nausea, vomiting, anxiety, to calm the heart, improve appetite, induce good sleep and even as a treatment for diabetes 12 . H. colchicifolia leaves, has not been scientifically validated previously eventhough it is used extensively in traditional medicine. The leaves may contain therapeutic potential like that of the corms. Therefore, this study aimed at investigating the potential biological activity of H. colchicifolia leaves and corms. Phytochemical analysis of H. colchicifolia has been done as the initial part of the study, and has been used to establish the mode of action of the extracts biological activity 13 .

Collection of Plant Material
Hypoxis colchicifolia was collected and identified using taxonomic keys by the School of Life Science, University of KwaZulu-Natal. The sampling site was located in Mooiriver, KwaZulu-Natal, South Africa with voucher specimens of the authenticated plant material deposited in the Ward Herbarium at UKZN (Westville campus) (Voucher number: Baijnathsn-01).

Preparation of Plant Material
Fresh as well as dried corms and leaves of Hypoxis colchicifolia were washed, cut and allowed to air dry. Plant material was then coarsely ground in an industrial grinder (RetschGmbh, West Germany), and stored in labeled Schott bottles in cool dark condition for further use.

Extraction of Plant Material
The fresh corms (150g), dried corms (20g) and leaves (20g) were extracted using different solvents (acetone, methanol, distilled water) at the ratio of 1:4 w/v, for 48 h on a rotary shaker and filtered using Whatman No. 1 filter paper. Filtrates were then evaporated using a Buchi rotary evaporator with resulting extract air dried further, with the aqueous extract evaporated at 40 0 C in a drying incubator. All extracts formed a solid, glass like extract that was stored in the dark at room temperature till required.

Alpha Amylase Inhibition Assay
Alpha amylase inhibition was tested using the method by Ranilla et al., 14 with minor modifications. Sodium Potassium Tartrate solution was made by adding 12 g of KNa 2 C 4 H 4 .4H 2 O to 8 mL of 2 M NaOH and heated till dissolved. Twenty milliliters of 96 mM 3,5 Dinitrosalicylic acid (DNS) solution was made in distilled water and was heated till dissolved. The DNS solution was then added to the Sodium Potassium Tartrate solution with the addition of 8 mL distilled water. This was allowed to stir in the dark overnight (±16 h). A 20 mM sodium phosphate buffer was made up with 6 mM NaCl. The extracts were suspended in the sodium phosphate buffer (1mg/mL concentration). Starch solution (1%) was made in sodium phosphate buffer. One milligram of 1% soluble starch solution was added to 1 mL of sample (200, 400, 600, 800, 1000 µg/mL) and was incubated for 5 min.
Journal of Natural Remedies | ISSN: 2320-3358 http://www.informaticsjournals.com/index.php/jnr | Vol 21 (3) | July 2021 Thereafter 1 mL of 1 unit/mL α-amylase solution made in sodium phosphate buffer was added and incubated for 3 min. DNS solution (1 mL) was added to the reaction mixture and the reaction was thereafter boiled for 15 min at 100 0 C. The samples were then cooled to room temperature and 9 mL of distilled water was added. The samples were then transferred to a 96-well plate and read at 540 nm. Absorbance values were converted into percentage Inhibition using the following equation:

Alpha Glucosidase Inhibition Assay
The α-glucosidase assay was conducted using the method by Ranilla et al., (2009) with minor modifications. A 50 µL sample (200, 400, 600, 800, 1000 µg/mL) was added to 50 µL of 0.1 M potassium phosphate buffer (pH 6.9) and 100 µL of 1 U/mL α-glucosidase enzyme solution (in 0.1 M potassium phosphate buffer, pH 6.9) was added. This was then incubated at 25°C for 10 min. Following pre-incubation, 50 µL of 5 mM p-nitrophenyl a-dglucopyranoside solution (in 0.1 M potassium phosphate buffer) was then added. This was further incubated at 25°C for 5 min. The control used was the buffer in place of sample and the blank was the buffer in place of the enzyme. The absorbance was read at 405 nm before and after incubation using a micro plate reader, with percentage inhibition calculated using the following equation:

Anti-hypertension (ACE Inhibition Assay)
The ACE inhibition assay was conducted according to Li et al., 15 and Chen et al., 16 with minor modifications. Twenty microliters of sample (200, 400, 600, 800, 1000 µg/mL) was suspended in sodium borate buffer, 50 µL of 5 mM HHL (in 0.1M sodium borate buffer) and 0.3 M sodium chloride (pH 8.3). This was then pre-incubated at 37ºC for 30 min. Thereafter 10 µL (1 U/mL) ACE solution was added to initiate the reaction. This reaction was incubated at 37ºC for 30 min.
One hundred microliters of 1 M HCl was added to stop the reaction and absorbance read at 492 nm. The sample blank was buffer in place of enzyme solution and the sample control buffer in place of sample.

Cytotoxicity Screening (MTT Assay)
Human embryonic kidney (HEK-293), breast cancer (MCF-7) and human lung cancer (A549) cell lines were obtained from the Department of Human Physiology at the University of KwaZulu-Natal, Westville campus and grown at 37°C in a humidified incubator under 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM). The 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the isolates. The MTT assay was conducted according to Dwarka et al.,17 with minor modifications. Briefly, cells (50 µL) (1x10¬2 cells/mL) as well as 50 µL DMEM were seeded in 96-well flat bottom plates and incubated for 24 h at 37°C in a humidified incubator under 5% CO2¬. Cells were then treated with 50 µL of sample extract prepared in 5% DMSO (7.8-1000 µg/mL) and incubated for 24 h. Camptothecin was used as the positive control. MTT reagent (20 µL, 5 mg/mL) was added to the cells and incubated (4 h at 37°C). Finally, 100 µL of DMSO was added to each well in order to solubilize the formazan salt formed. The absorbance was read at 570 nm on a micro plate spectrophotometer (Multiscan Go, Thermo Scientific) and the percentage viability determined using the formula:

Statistical Analysis
Results were analyzed by ANOVA (Graph Pad Prism software, San Diego, CA, USA). All analyses were done in triplicate, mean±standard deviation was calculated. IC 50 was also calculated using Graph Pad Prism. The lower the IC 50 concentration, the more potent the extract as a therapeutic.

ACE Inhibition
The IC 50 for ACE inhibition in descending order are as follows: dried leaf acetone (705.6 µg/ml), fresh corm aqueous (691.2 µg/ml), dried corm acetone (628.2 µg/ml), dried corm aqueous (569.4 µg/ml), dried leaf methanol (542.6 µg/ml), dried leaf aqueous (537 µg/ml), fresh corm acetone (503 µg/ml), fresh corm methanol (439.7 µg/ml) and dried corm methanol (368.2 µg/ml) (Figure 3). The IC 50 of the positive control captopril was 442.5 µg/ml.The methanol extract of H. colchicifolia dried corms has the lowest IC 50 , denoting optimal dosage for ACE inhibitory potential and the acetone extract of dried leaves had the highest IC 50 value. Only the methanol extract of fresh and dried corms was more effective than that of the positive control, captopril. There were no significant differences between the results of the following extracts: FCA and FCAQ, FCA and DLM, FCA and DCM, FCM and FCAQ, FCM and DCM, FCAQ and DLM, DLA and DCAQ, DLM and DCM, DCM and DCAQ. The rest of extracts tested had a significant difference (p<0.0001) with each other and the positive control. This mainly denotes no significant difference between fresh corms extracts; no significant difference in between the methanlic extracts of fresh and dried corms. Duncan et al., 23 found that aqueous and ethanolic extracts of leaves and roots of H. colchicifolia tested for ACE inhibition produced poor inhibition of between 4-37% inhibitions, with both leaf extracts having a greater inhibition than that of root extracts. Arhin et al., 24 showed a greater ACE inhibitory potential of the methanolic extracts of the leaves of Tulbaghia acutiloba with inhibition activity of 76.66 ± 1.65 (IC 50 154.23 μg/mL).

MTT Cytotoxicity
The IC 50 for HEK-293 inhibition in descending order are as follows: dried leaf methanol (14.16 µg/ml), dried leaf aqueous (11.35 µg/ml), dried leaf acetone (9.02 µg/ml), dried corm aqueous (7.99 µg/ml), fresh corm methanol (7.98 µg/ml), dried corm acetone (7.96 µg/ml), dried corm methanol (7.93 µg/ml), fresh corm aqueous (7.34 µg/ml) and fresh corm acetone (5.39 µg/ml) (Figure 4). The IC 50 of the positive control camptothecin was 9.06 µg/ml.The acetone extract of H. colchicifolia fresh corms produced the greatest cell inhibition and the methanol extract of dried leaves had the lowest cell inhibition in HEK-293 cell line. All extracts tested showed no significant difference when compared to each other and the control. A study by Madikizela and McGaw 25 showed that the corm extracts of H. colchicifolia had an LC 50 values of 2.48, 0.89 and 0.98 mg/mL against Vero monkey kidney cells for aqueous, ethanol and acetone extracts respectively. A study by Madikizela and McGaw (2018) showed that the corm extracts of H. colchicifolia had an LC 50 values of 2.48, 0.89 and 0.98 mg/mL against Vero monkey kidney cells for aqueous, ethanol and acetone extracts respectively.
The IC 50 for MCF-7 inhibition in descending order are as follows: fresh corm acetone (9.51 µg/ml), fresh corm aqueous (7.49 µg/ml), dried corm aqueous (7.41 µg/ ml), fresh corm methanol (7.28 µg/ml), dried leaf acetone (7.19 µg/ml), dried corm acetone (4.52 µg/ml), dried corm methanol (4.34 µg/ml), dried leaf aqueous (3.83 µg/ml), and dried leaf methanol (3.24 µg/ml) ( Figure 5). The IC 50 of the positive control camptothecin was 8.44 µg/ml.The methanol extract of H. colchicifolia dried leaves produced the greatest cell inhibition and the acetone extract of fresh corms had the lowest cell inhibition in MCF-7 cell line. All extracts examined showed no significant difference when compared to each other and the control. In a cytotoxicity screening of African medicinal plants, Steenkamp  The IC 50 for A549 inhibition in descending order are as follows: dried leaf aqueous (280 µg/ml), fresh corm aqueous (270.3 µg/ml), fresh corm acetone (228.7 µg/ml), fresh corm methanol (215.9 µg/ml), dried corm methanol (118.9 µg/ml), dried leaf acetone (95.65 µg/ml), dried corm acetone (87.07 µg/ml), dried leaf methanol (68.68 µg/ml) and dried corm aqueous (32.22 µg/ml) ( Figure 6). The IC 50 of the positive control camptothecin was 304.2 µg/ml. The aqueous extract of H. colchicifolia dried corms has the highest cell inhibition and the aqueous extract of dried leaves had minimal inhibition in A549 cell line. These results indicate that the extracts are toxic to cancerous cells while not producing a drastic decrease in normal cells. All extracts tested showed no significant difference when compared to each other and the control. This is in opposition to studiesby Madikizela and McGaw 28 who found that aqueous extracts of corms to be least toxic and had the highest IC 50 (2480 µg/mL) in non-cancerous Vero African monkey kidney cells. However, when tested in A549, CaCo-2, HELA and MCF 7 in different solvents (acetone, ethanol, hot and cold water), had an IC 50 ranging from 50-251.95 µg/mL. Hypoxis colchicifolia has anticancer potential could be due to glycoside hypoxoside and rooperol activity 26 . In a study by Steenkamp and Gouws 29 , corms of Hypoxis were found to be non-cytotoxic against prostate cancer cells, breast cancer cells and non-malignant breast cancer cell lines at a concentration of 50 µg/mL.

Conclusion
The fresh corms acetone extract was effective in producing anti-diabetic effects with the dried corm methanolic extract being active in hypertension suppression. Methanol extracts of dried leaves were successful in inhibiting cancerous cell lines while remaining nontoxic to noncancerous cell lines. This study shows that different parts of the plant have different capabilities as a therapeutic and cannot be used interchangeably. Although H. colchicifolia has potential to act as a therapeutic, further in vivo studies need to be conducted.