Wound Healing, Antimicrobial and Antioxidant Properties of Clinacanthus nutans (Burm.f.) Lindau and Strobilanthes crispus (L.) Blume Extracts

Clinacanthus nutans is known to be an anticancer and antiviral agent, and Strobilanthes crispus has proven to be an antidiuretic and antidiabetic agent. However, there is a high possibility that these plants possess multiple beneficial properties, such as antimicrobial and wound healing properties. This study aims to assess the wound healing, antioxidant, and antimicrobial properties of Clinacanthus nutans and Strobilanthes crispus. The Clinacanthus nutans and Strobilanthes crispus leaves were dried, ground, and extracted with ethanol, acetone, and chloroform through cold maceration. In a modified scratch assay with co-incubation of skin fibroblast and Methicillin-resistant Staphylococcus aureus, Clinacanthus nutans and Strobilanthes crispus extracts were assessed for their wound healing potential, and the antimicrobial activities of Clinacanthus nutans and Strobilanthes crispus extracts were performed on a panel of Gram-positive and Gram-negative bacteria on Mueller–Hinton agar based ona disc diffusion assay. To assess for antioxidant potential, 2,2-diphenyl-1-picrylhydrazyl (DPPH), total phenolic and total flavonoid assays were conducted. In the modified scratch assay, Clinacanthus nutans extracts aided in the wound healing activity while in the presence of MRSA, and Strobilanthes crispus extracts were superior in antimicrobial and wound healing activities. In addition, Strobilanthes crispus extracts were superior to Clinacanthus nutans extracts against Pseudomonas aeruginosa on Mueller–Hinton agar. Acetone-extracted Clinacanthus nutans contained the highest level of antioxidant in comparison with other Clinacanthus nutans extracts

In-vitro Study of Cytotoxicity and Apoptosis Effects of Clinacanthus nutans (Burm.f.) Lindau Extracts on Colorectal Cancer Cell Lines, HT-29 and HCT-116

Cancer is one of the leading causes of mortality in Malaysia, with colorectal cancer as the most common cancer in males and the second most common cancer in females. Colorectal cancer causes high mortality as it is hardly detected during the early stage yet has a 15-year window of intervention. Hence, the discovery of the chemoprevention method is important to delay carcinogenesis and prevent the recurrence of cancer. Clinacanthus nutans (Burm.f.) Lindau (C. nutans), from Acanthaceae family, is known as Sabah Snake Grass or ‘Belalai Gajah’ in Malaysia. It is popular in Southeast Asia for its medicinal properties such as anticancer, anti-inflammation and antiviral properties. Therefore, this study aimed to determine the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and anticancer properties of C. nutans leaves extracts on human colorectal cancer cell lines, HCT-116 and HT-29 in dose- and time-dependent manners. The ground C. nutans leaves were extracted with methanol, chloroform, and acetone for 30 minutes and 24 hours, respectively. The TPC and TFC were determined spectrophotometrically using the Folin-Ciocalteu and aluminium chloride colourimetric methods. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay was used to determine the antioxidant activity. The antiproliferation and apoptotic capabilities were studied using MTT and apoptosis (Annexin V-FITC and PI staining method) assays, respectively. C. nutans methanol extracts and 24 hours of extraction time resulted in higher extraction yield (7.65g ± 0.13) than its counterparts. Acetone extracted C. nutans at 30 minutes and 24 hours resulted in the highest TPC values of 3.06 mg GAE/g and 3.18 mg GAE/g, respectively. On the other hand, methanol extracted C. nutans at 30 minutes and 24 hours exhibited the highest TFC (0.49 mg QE/g and 0.50 mg QE/g, respectively) and strongest antioxidant activity (IC50 values at 24.25 μg/mL and 19.67 μg/mL; AAEAC at 22.14% and 27.31%, respectively) than chloroform and acetone extracts. The methanol extracts were selected for in vitro study. HCT-116 cells revealed higher antiproliferative effects than HT-29 cells using methanol extracted C. nutans at 30 minutes when treated for 24 hours and 72 hours. On the contrary, HCT-116 cells exhibited lower antiproliferative effects when treated for 48 hours. Methanol extracted C. nutans at 24 hours exhibited stronger antiproliferation activity when treated on HT-29 cells with less cytotoxicity to normal cells for 24, 48, and 72 hours. In addition, a low dose of 250 μg/mL methanol extracted C. nutans with 24 hours extraction time induced early and late apoptosis in HCT-116 and HT-29 cells after treatment for 72 hours. The methanol extract increased the late apoptosis of HCT-116 cells from 18.88% to 35.66% when treatment from 24 hours to 72 hours, respectively. The late apoptotic rate of HT-29 cells was increased from 14.28% to 20.67% when treatment was prolonged from 24 hours to 72 hours, respectively. In conclusion, methanol extracted C. nutans that underwent 24 hours extraction contained high TFC, exhibited high antioxidant activity, and able to inhibit the early and late stages of colorectal cancer cell growth by triggering apoptosis in dose- and time-dependent manners. The findings suggested that C. nutans possessed anticancer properties against colorectal cancer cell lines, HCT-116 and HT-29 through the apoptosis pathway. The findings provided new perspective for the future study to prove C. nutans as an alternative treatment against early and late stages colorectal cancer

Antioxidant, Antimicrobial and Wound Healing Properties of Clinacanthus nutans (Burm. f.) Lindau and Strobilanthes crispus (L.) Blume Extracts

Clinacanthus nutans (C. nutans) and Strobilanthes crispus (S. crispus) both are well-known for their specific beneficial properties against different diseases. C. nutans is known to be anticancer and antiviral, especially against colon cancer, varicella-zoster virus, and herpes simplex virus. S. crispus has proven to be anti-diuretic and antidiabetic by multiple studies. In the previous studies, S. crispus extract showed promising results, capable of aiding in the wound healing process. Since C. nutans was commonly available as a balm in the traditional market, there is a high possibility that it contains antimicrobial and wound healing properties as well. Thus, this study aimed to confirm the medicinal benefits of C. nutans and S. crispus, specifically antimicrobial and wound healing properties. The C. nutans and S. crispus leaves were extracted with different polarity solvents; ethanol, acetone, and chloroform, through cold maceration and kept refrigerated at 2-8°C in the dark. The presence of phytochemicals, such as alkaloids, flavonoids, and saponin in the extracts were screened. These extracts were then assessed for their antioxidant potential by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay using ascorbic acid as standard; determination of Total Phenolic Content (TPC) and Total Flavonoids Content (TFC) using Gallic Acid and Rutin as standard, respectively. Disk Diffusion Assay was performed to determine their antimicrobial properties. Modified scratch assay by co-incubating skin fibroblast with Methicillin-resistant Staphylococcus aureus (MRSA) and treated with the C. nutans and S. crispus extracts to determine their wound healing effects. These extracts were observed at 1, 3, 6, 10, 24, and 48 hours to investigate the cell migration activity depicting the closing up of wound. The phytochemical profiles for both C. nutans and S. crispus extracts were similar, except the C. nutans extracts have a higher saponin level compared with S. crispus extracts. The C. nutans acetone extract contained the highest level of antioxidant potential and TPC than the other C. nutans extracts. However, the ethanol extract of C. nutans showed the highest TFC, which was corresponded to the flavonoid content obtained from the phytochemical screening. The highest TPC value was recorded by the S. crispus ethanol extract, while the TFC test revealed similar pattern results among these S. crispus extracts. The C. nutans extracts showed a zone of inhibition of 14 to 16 mm when treated on Ps. aeruginosa, while S. crispus extracts showed distinctive zone of inhibition range between 11 to 15 mm on Ps. aeruginosa. There was no positive result observed when the extracts were treated on other bacteria. Fibroblasts were sensitive to acetone extracts, and S. crispus extracts were proven to have higher toxicity during the MTT assay. During the scratch assay with extracts only, C. nutans extracts showed wound healing properties, whereas S. crispus extracts seemed to be delayed the wound healing activity. However, when MRSA was added for the co-incubation, S. crispus extracts showed inhibition against the growth of MRSA and aided the wound healing activity, but this situation did not demonstrate in C. nutans extracts. The C. nutans and S. crispus extracts exhibited strong antioxidant and antimicrobial properties. C. nutans extracts aids in the wound healing activity, but if MRSA was involved, S. crispus extracts were capable of inhibiting the growth and aiding in the wound healing activity