Alkaloid, Phenolic profiling and Biological Activities of Caralluma russeliana Methanol Extracts from Al Baha region, Saudi Arabia

Introduction

Cancer and disease due to microbes are one of the major causes of mortality. The drug candidates currently used for cancer therapy have developed resistance and toxicity despite of the advancement in technology and drug development¹. Moreover, bacterial infections occur very commonly among cancer patients owing to weak immunity which results from radiotherapy, chemotherapy, prolonged hospitalizations, malnutrition, and invasive procedures². The overuse of antibiotics is the main drivers to drug resistant microbial pathogens, particularly, methicillin resistant S aureus which mostly prevail in hospitals³. Natural products based drugs or extracts are safer compared to synthetic drugs and are in used for the cure of different disease since ancient time, due to their lower side effects and better compatibility⁴. Natural products display diverse biological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial, antitubercular, and antipyretic^5,6 that are  attributed to the plant secondary metabolites such as flavonoids, alkaloids, phenolics, tannins, terpenoids, and glycosides present in plant extract.

Caralluma (fam. Asclepiadaceae), constitute around 200 genera and 2500 species which is widely distributed in Europe, Africa, South Africa, Canary Island and Arabian Peninsula. Carulluma species are important part of traditional system of medicine in various countries. C. fimbriata is medicinally used in pain, inflammation, diabetes and appetite suppressant, C. tuberculata as food and for the treatment of fever, leprosy and diabetic, while C. attenuata for migraine and diabetes. Caralluma species (C.fabricata, C. edulis, C. tuberculata and C. umbellate, C. laciantha, C. stalagmifera, C. arabica) possess important pharmacological properties like antidiabetic, antiinflammatory, anticancer, antimicrobial, antioxidant and antigastric⁷.

Caralluma russeliana, occupy the rocky mountains of Al Baha region, situated on southern part of Saudi Arabia (Figure 1). This small, erect and fleshy, perennial herbs have been used in folk medicine in the treatment of cancer, diabetes, inflammation, fever, tuberculosis, skin rashes, snake bite and scorpion bite^8-12. Recent studies have reported that C. russeliana stem extracts possess significant antidiabetic potential and caused reduction in complications such as body weight, lipid profile, AST, ALP in STZ induced rats¹³. These medicinal values are attributable to the phytoconstituents particularly pregnane glycosides, russeliosides A–D, flavone glycosides, luteolin, which have been isolated from this plant^14-16. The above studies encouraged us to explore this plant from AL Baha region, KSA. Therefore, in this work, we investigated the antioxidant, anticancer and antimicrobial studies of C. russeliana, which is the first study from this place.

Figure 1: Caralluma russeliana plant17 Click here to View figure

Experimental

Extraction

The aerial parts of Caralluma russeliana was collected from Albaha region, KSA and authenticated by taxonomist. The plant was Soxhlet extracted using methanol as a solvent to afford the methanol extract, dried completely under vacuum and kept in refrigerator. The extract was then screened for its phytochemical analysis, antioxidant, antimicrobial, anticancer and HPLC analysis

Phytochemical Analysis

The amount of total phenolic (TPC), total flavonoid (TFC), total tannins and total alkaloids was quantified using method of Folin–Ciocalteu colorimetric¹⁸,Chang et al¹⁹, Broadhurst and Jones²⁰ and Shamsa et al²¹.

Antioxidant activity

The extract was screened for antioxidant potential at Regional Center for Mycology and Biotechnology (RCMB) at Al- Azhar University by DPPH, ABTS, NO, H₂O₂ and ferric reducing power method as previously reported²².

Anticancer activity

The extract was screened for cytotoxicity by MTT as previously reported²³ against skin (A-431), lung (A549), colorectal (HCT-116), hepatocellular (HepG2), prostate (PC-3) and breast (MCF-7) and a normal healthy lung fibroblast (MRC-5), which were procured from American Type Culture Collection. Doxorubicin was used for comparison.

Antimicrobial activity

The plant methanolic extract was evaluated against different bacterial and fungal strains at Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Two Gram positive strains (Staphylococcus aureus and Bacillus subtilis) and two Gram negative bacterial strains (Escherichia coli, Proteus vulgaris) were taken and the extract was tested against them. The plant extract was also tested against two fungal strains (Aspergillus fumigatus, Candida albicans) using a modified well diffusion method. The extract was dissolved in DMSO to prepare solution of 10 mg/mL as stock solution. The agar plates were spread with bacterial and fungal strains and 100 μL of extract solution was added in well of each plate. The plates were kept at 37 ⁰C for 24-48 hrs. After incubation microorganism were grown on to the plates and zone of inhibition was measured. For this experiment, DMSO was taken as negative control, Gentamycin was taken as positive control for bacterial strains and Ketoconazole was taken as positive control for fungal strains at a dose of 1 mg/mL, 100 μL of each control was added in the well. The microbial strains sensitive towards the extract was subjected to determine MIC by using broth dilution method

HPLC analysis

The phenolics and alkaloids in the extract was analyzed by HPLC using Agilent 1100 instrument according to method of Lin et al²⁴

Results and Discussion

Phytochemical Analysis

C. russeliana contains important classes of compounds like phenols, flavonoids, tannins and alkaloids as exhibited by the phytochemical analysis of its methanol extract. As illustrated in Figure 2, the extract contains 16.75 mg/gm total phenolic content, 7.41 mg/gm total flavonoid, 1.29 mg/gm total tannin and 2.96 mg/gm total alkaloid content (TAC). These different classes of compounds in the extract were further assayed by HPLC for determination of alkaloid and phenolics.

Figure 2: Phytochemical Analysis of methanol extract of Caralluma russeliana. Click here to View Figure

HPLC Analysis of the methanol extract

The HPLC analysis of methanol extract of Caralluma russeliana was performed to detect different alkaloids and phenolics (figure 3, 4). The different alkaloids namely Evodiamine, berberine, protopine, camptothecin and cycloclavin whereas different phenolics namely chlorogenic, catechol, syringic acid, p-coumaric, pyrogallol, gallic acid and salicyclic acid were observed in different concentration at different retention time (RT). Among the identified alkaloids, cycloclavine was the major components (9.16 μg/mg) followed by camptothecin (6.33 μg/mg) and berberine (4.13μg/mg). Evodiamine was present in lower concentration (3.22 μg/mg) while protopine in a fairly low concentration (0.79 μg/mg). In case of identified phenolics, p-coumaric acid, gallic acid and syringic acid was the major with concentration of 11.69 μg/mg, 6.14 μg/mg and 4.0 μg/mg, respectively. Chlorogenic, syringic, pyragallol and salicyclic acid were present in lower concentration of 3.02, 4.0, 3.88, and 2.10 μg/mg, respectively whereas catechol was less than 1% (table 1, 2). This analysis indicates that the extract contains biological active alkaloids and phenolics.

Figure 3: Alkaloids identified in the methanol extract by HPLC analysis. Click here to View figure

Table 1: Alkaloids identified in methanol extract of C. russeliana Click here to View table

Figure 4: Phenolics identified in the methanol extract by HPLC analysis Click here to View figure

Table 2: Phenolics identified in methanol extract of C. russeliana. Click here to View table

Antioxidant activity

The antioxidant activity of the methanol extract of C. russeliana as measured by DPPH, ABTS, NO, H₂O₂ and FRAP are depicted in Table 3 as IC₅₀ in μg/mL. The extract exhibited concentration dependent scavenging activity. The extract strongly quenched DPPH with IC₅₀ of 119.17 μg/ml, compared to BHT (22.14 μg/ml) and Ascorbic acid (10.21 μg/ml). The ABTS, NO and H₂O₂ scavenging by the extract was found to be 155.71 μg/ml, 223.40 μg/ml and 184.40 μg/ml, respectively. The FRAP of the extract (203.48 μg/ml) was significantly lower than BHT (50.35 μg/ml) and ascorbic acid (20.89 μg/ml). Based on these observations, the constituents identified in extract have the ability to scavenge DPPH^• and quench ABTS^+• and reduce Fe³⁺ through various mechanisms.

Table 3: Antioxidant activity (IC₅₀) of methanol extract of Caralluma russeliana.

Extract	DPPH (μg/ml)	ABTS  (μg/ml)	NO (μg/ml)	H2O2 (μg/ml)	FRAP (μg/ml)
Methanol	119.17 ± 5.97	155.71± 9.59	223.40± 5.77	184.40 ± 22.39	203.48 ± 5.41
BHT	22.14 ± 1.82	50.69 ± 1.25	19.92± 2.21	116.18 ± 4.58	50.35± 4.99
Ascorbic acid	10.21 ± 0.77	10.66 ± 0.89	17.95± 2.24	14.77 ± 0.69	20.89 ± 1.25

Results are mean of three experiments ± SD.

Oxidative stress is the imbalance between reactive oxygen species (ROS) and antioxidative defence system.  ROS hampers the antioxidant system in the cells that damage biomolecules like DNA, RNA, etc. Constituents derived from plants play a crucial part in the treatment of many diseases by targeting different mechanisms. These natural product protect the cells from oxidative stress causing amelioration of many diseases. C. russeliana have been traditionally used for various ailments which may attributable to important phytoconstituents such as alkaloids and phenolics identified in HPLC. Evodiamine, chemically (+)-(S)-8,13,13b,14-tetrahydro-14-methylindolo [2′,3′:3,4]pyrido[2,1-b]quinazolin-5(7H)-one), is a quinozole alkaloid that has protective role in acute kidney injury and decreases the blood urea and creatinine levele in LPS induced rats²⁵. Berberine, an isoquinoline alkaloid has been used in Ayurvedic, Persian and Chinese traditional medicine since time immemorial. This alkaloid can strongly scavenge free radicals and lessen oxidative stress throughPI3K/Akt/Bcl2/Nrf2/SIRT1 and AMPK pathways²⁶. Chlorogenic acid, an ester of caffiec acid and quinic acid is a well known antioxidant which scavenge oxygen and nitrogen species. Due to its polyhydroxy structure, it can activate Nrf-2/HO-1 antioxidant signalling pathways and enhances antioxidant ability by regulating expression of various genes²⁷. Syringic acid is chemically known as 4-hydroxy-3,5-dimethoxy benzoic acid that hamprs different oxidative stress markers, and inhibit the oxidation of LDL and NADPH oxidase²⁸. Gallic acid bearing three hydroxy can scavenge free radical strongly which bestow protection from oxidative stress²⁹. p-coumaric acid is a phenolic compound with capability to scavenge free radicals. It can inhibit 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) induced ROS generation and can increase the expression of antioxidant genes³⁰. Salicylic acid generates ROS and acts as radical scavenger therefore is involved in cell oxidative stress³¹. The different mechanisms of these alkaloids and phenolics in the methanol extract of C. russeliana might be imparting the antioxidant protection against oxidative stress.

Anticancer activity

The anticancer activity of the methanol extract of C. russeliana as screened by MTT protocol against a panel of six human adenocarcinomas is illustrated in table 4. The six adenocarcinomas, namely skin (A-431), lung (A549), colorectal (HCT-116), hepatocellular (HepG2), prostate (PC-3) and breast (MCF-7) while a normal healthy lung fibroblast were procured from American Type Culture Collection. For comparison, doxorubicin was used as a positive control. The methanol extract demonstrated moderate to promising antiproliferative activity towards most of the tested cancer cell lines. Among the tested cell lines, hepatocellular (HepG2) and lung (A549) were the most sensitive cell lines towards the extract. The extract significantly block proliferation with IC₅₀ 24.37 μg/mL in HepG2 and 26.84 μg/mL in A549 cells, compared to Doxorubicin which inhibited proliferation with IC₅₀ 0.37 μg/mL and 1.01 μg/mL, respectively. The extract was also cytotoxic towards colorectal HCT-116 and skin A-431 with IC₅₀ 49.07 μg/mL and 40.78μg/mL, respectively whereas moderately inhibited prostate PC-3 cells with IC₅₀ 57.11 μg/mL.  The extract displayed mild cytotoxicity on breast MCF-7 cells.  However, the anticancer activity of the extract was lower than the positive control, Doxorubicin. From safety point of view, the extract was also tested on normal lung cells (MRC-5) and found to be non-toxic on normal cells.

Cancer, the second global cause of mortality is increasing at an alarming rate, therefore prevention and treatment of cancer requires urgent startegies³². Bioactive molecules present in different plant extracts have emerged as potential anticancer agents. In plants, phenolics and alkaloids have gain considerable interests in drug discovery as they have provided many drugs for various treatment³³.  These bioactive agents regulates apoptotic proteins, oxidative stree, or various transcription signalling pathways³⁴. Berberine are known to block proliferation by cell cycle regulation, cell autophagy, inhibits cell invasion, metastasis, downregulates/upregulates vaious metastasis and apoptosis proteins by suppressing signalling pathways like C-myc, cyclin-D1, and MMP-3 expressions and inhibition of PI3K/AKT/mTOR signaling^35,36. Camptothecin is a quinoline based pentacyclic monoterpenoid indole alkaloid which is a potent topoisomerase II inhibitor and NRF2 inhibitor. This natural product by semi synthesis has afforded many more potent anticancer drugs like Irinotecan, Topotecan or Camtosar which have been granted by WHO for use in sevaral cancers like ovarian, brain, uterine, lung, and colon^37,38. Evodiamine, acts as modulator of topoisomerase I inhibitor, NF-κB, Bcl2, MAPK, suppress H60 expression in KATO III stomach cancer and HeLa S3 uterine cancer and induces apoptosis in many cancer cells like breast, prostate, cervical, melanoma, leukemia, colon and lung^39-41. Previous study reports that gallic acid induce BIK-BAK/BAK, caspase-3 and 9 and Bcl-2 pathways involved in apoptosis, and regulate oxidative stress⁴². Catechol has been known to have suppressing action on ERK2/c-Myc signals in lung cancer, EMT‑related proteins via AMPK/Hippo signals in pancreas cancer⁴³.  Syringic acid acts as anticancer agent by regulating various expression like Cdk4 and Cdk6 of cell cycle and pre and proapoptotic genes like Bax, Bak, Bcl2, and FLIP²⁸. p-coumaric acid is capable of inhibiting cell proliferation and migration and triggers apoptosis in lung and colon cancer⁴⁴. Chlorogenic acid is an effective natural anticancer drug approved by the China Food and Drug Administration. It is a safe differentiation inducer for solid tumors, inhibits HIF-1α/AKT pathway, inhibits MAPK/ERK activation via ROS overproduction and induces DNA damage and formed topoisomerase-DNA complexes involved in apoptosis and a chemosensitizing chemotherapy agent^45-48.  Pyrogallol, is known to have antitumor effects in breast and colon cancers via miR-134 activation-mediated S-phase arrest and inhibition of PI3K/AKT/Skp2/cMyc signaling^49,50. Salicylic acid is known to trigger ER stress-induced apoptosis via Akt/mTOR/AMPK pathways and upregulation of nitric oxide production⁵¹. These biactive may be attributable to the anticancer activity by the methanol extract of C. russeliana.

 Table 4: Anticancer activity (IC₅₀, μg/mL) of the methanol extract of Caralluma russeliana

Cell lines	Origin	IC50 (μg/mL)
Cancerous cells		Extract	Doxorubicina
breast	MCF7	111.21±3.38	0.5 ± 0.03
Hepatocellular	HepG2	24.37±1.01	0.37±0.02
Colorectal	HCT-116	49.07±2.40	0.49±0.04
Lung	A549	26.84±2.14	1.01±0.20
Prostate	PC3	57.11±0.86	3.34±0.31
Skin	A-431	40.78±2.59	11.26±0.54
Normal cells
Lung	MRC-5	521.65±25.32	3.13±0.29

 

Antimicrobial activity

The methanol plant extract was tested against different bacterial and fungal strains using disc diffusion method. The results of antimicrobial activity are shown in table 5. The plant extract was active against the bacterial strains and inactive against the tested fungal strains. The extract was active against one Gram positive bacterial strain S.aureus and one Gram negative bacterial strain P.vulgaris and showed zone of inhibition 9.86 mm and 10.90 mm respectively. When results were compared with the standard compound Gentamycin, the extract displayed mild antimicrobial activity. The plant extract was also evaluated for its MIC, against these two bacterial strains and showed MIC 3300 μg/mL and 1666.66 μg/mL against S.aureus and P.vulgaris, respectively.

Table 5: Antimicrobial activity of the methanol extract showing zone of inhibition (mm) and MIC (μg/mL).

Test Samples	Bacterial Strains				Fungal Strains
	S.aureus ATCC25923	B.Subtilis NRRLB-543	E.coli ATCC25922	P.vulgaris ATCC13315	A.fumigatus ATCC13073	C.albicans ATCC10231
Extract	9.86±1.22	NA	NA	10.9±1.12	NA	NA
Positive control	24.4±2.28	26.63±3.12	26.73±3.28	25.46±3.18	17.33±1.45	20.56±2.04
MIC	3300	NT	NT	1666.66	NT	NT

 

MIC: Minimum Inhibition Concentration (μg/mL); NA: Not Active; NT: Not tested

Antimicrobial resistance is a major burden which occurs when drugs acquire resistance towards many microbes like bacteria and fungi. As drug resistance is the main hurdle in infectious diseases, control of infections has been difficult. Natural products or their plant extracts are good choice for the prevention and treatment of infections⁵². Natural products are reservoir of antimicrobial drugs, for instance, Penicillin a notable antibiotic, Lysergic acid diethylamide, Cabergoline, Streptomycin, Gentamycin, Polymyxcin, Daptomycin, and so on have been isolated from bacteria and fungi⁵³. In the present work, identified natural products are endowed with antimicrobial property. Berberine displayed significant antimicrobial effect by inhibtiing antibiotic resistant microbes, damage cell wall and cell membrane of methicillin resistant S. aureus (MRSA) and E. coli, inhibit biofilm formation, supress protein synthesis and bacterial division⁵⁴. Evodiamine have antibacterial effect by inhibiting the relaxation of plasmid DNA while bactericidal against K. pneumoniae⁵⁵. Chlorogenic acid has displayed antifungal activity against fluconazole resistant Candida species by mitochondrial depolarization, ROS production and  phosphatidylserine externalization⁵⁶. Catechol has been shown to have antifungal effects on Colletotrichum circinans fungus and possess antibacterial effect⁵⁷. Syringic acid possess antimicrobial effect against various Gram positive and Gram negative bacteria. It could inhibit the growth of MRSA, Cronobacter sakazakii, E. coli, and influenced cell membrane permeability by proton influx and ion leakage⁵⁸. Salicyclic acid could inhibit the growh of many fungal pathogens like Botrytis cinerea, P. expansum and R. stolonifer via intracellular disorganization, lipid damage and outflow of pathogen’s proteins⁵⁹. Also, salicyclic acid microcapsules ruptures cell wall and cell membranes of E.coli and S. aureus therefore possess significant antibacterial potential⁶⁰. These mechaism of the isolated compounds may be responsible for the antimicrobial activity exerted by the methanol extract of C. russelliana.

Conclusion

In conclusion, the results of the present study, to some extent are in line with the traditional use of this plant as antioxidant, anticancer and antimicrobial agents. The methanol extract of Caralluma russeliana quenched DPPH, ABTS, NO and H₂O₂ radical significantly thus have antioxidant property. The extract was also found to be active against all the tested cancerous cell lines, among which HepG2 and A549 were the most sensitive in exerting toxicity by the extract. Also, the extract was active against S. aureus and P. vulgaris as observed by the antimicrobial assay. These biological activities might be due to presence of evodiamine, berberine, camptothecin, chlorogenic acid, syringic acid and p-coumaric acid which are known for their mode of action as antimicrobial and anticancer agents triggered by oxidative stress. Studies for the isolation and structure elucidation of anticancer, antimicrobial and antioxidant active constituents from the methanol extract of C. russeliana are in progress which could provide leads for the treatment of these illnesses in future.

Acknowledgment

The author gratefully acknowledges Deanship of Scientific Research (DSR), Albaha University for financial support (project grant number 1442/9) and chemistry department, Albaha University for the facilities.

Conflicts of Interest

There are no conflict of interest.

Funding Sources

The work is funded by the Deanship of Scientific Research at Albaha University, grant number 1442/9.

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