INTRODUCTION:

Embelia ribes Burm F is a medicinal woody plant that comes under the Myrsinaceae family. It is also called false black pepper or Vidanga. The Embelia ribes fruits are blackish brown in color with a size of 2.4- 4.00 mm, sub-globular shape, wrinkled textured, distinct odour, and astringent taste with a slight pungency. In herbal preparations, Embelia ribes roots, berries, and leaves are used¹.

E. ribes is one of the 32 medicinal plants that is marked by the National Medicinal Plant Board by Govt. of India in the ‘Priority Species List’ for cultivation and also included in Indian Pharmacopoeia. Embelin an orange color crystalline powder is the major active constituent of Embelia ribes having IUPAC name 2,5-dihydroxy-3-undecyl-1,4-benzoquinone with molecular formula C₁₇H₂₆O₄ and molecular weight of 294.391g/mol. The Embelin (Fig. 1) is responsible for most of the biological activity.

It is used in a variety of formulations and is a part of both traditional herbal medicine and in Indian system of medicine and homeopathy (ISM&H)².Chemically it consists of Embelin (2.3%), quercitol (1.0%), embelinol, embelia ribyl ester, and embeliol, an alkaloid christembine; tannin; vilangin. Resinoid, fixed oil, and traces of volatile oil are also present³.

Figure 1: Structure of Embelin

Table 1: Properties of Embelin

Melting point	142.50˚ C
Appearance	Orange solid crystal
Log p	4.34
Density	1.1 g/cm³
Solubility	Soluble in hot organic solvents like DMSO and alkali hydroxide solution. Very slightly soluble in petroleum ether and insoluble in water⁴.
Storage	Store at 20°C
Reaction	1. Embelin undergoes oxidation, and to a lesser extent thermal deterioration. 2. When Embelin is held in alcohol (methanol or ethanol) in the presence of acid, it goes through an etherification process and produces the corresponding Embelin ethers⁵.

ISOLATION TECHNIQUES OF EMBELIN FROM EMBELIA RIBES FRUITS:

There are several ways for the isolation of Embelin. Indian herbal pharmacopeia suggested a hot extraction method by refluxing the Embelin fruit powder in n-hexane for six hours and evaporating the extract in a mixture of chloroform and ethanol for recrystallization⁶. By initially extracting the fruits in ethyl acetate at room temperature for 72 h, concentrating the extract, and then using chromatography on colloid columns, Daniel et al., were able to isolate Embelin from E. Schipper⁷. Sekaret al., extracted the Embelin fruit powder by cold maceration method using n-hexane, and the concentrated extract was chromatographed on a colloid column. Orange-colored Embelin was produced after recrystallizing using aromatic hydrocarbon extraction (yield: 0.325% w/w)⁸. Latha et al., isolated Embelin by using a different approach usinga microwave-assisted extraction method using acetone and concluded that non-polar solvents like n-hexane and dichloromethane didn’t give a good yield of Embelin powder⁹. Mr. Kilambi Pundarikakshudu typically uses simple extraction with inhalation general anesthesia for 20min, followed by washing the residue with rock oil ether, to perform gravimetric determination of Embelin from E. ribes fruits. He conducted a study in which Embelin was isolated using entirely different procedures which resulted in making a less time-consuming technique. The isolated Embelin was characterized for melting point, UV, FTIR, mass, and 1H magnetic resonance spectra¹⁰. Embelin isolated with chloroform and ether on UV analysis showed lambda max at 305 nm, lR spectrum showed bands at 3302.1cm^-1 (O-H stretching), asymmetric and symmetric stretching at 2916.37cm^-1 and 2846.93cm^-1respectively. 1612.49cm^-1 (C=O stretching), 856.39cm^-1 (C- H out of plane), and 1458.18cm^-1 (C-H bending of-CH group). The spectrum showed an M+, m/z peak at 296nm. To confirm the purity of Embelin 1H-NMR was performed (400 rate, CDCl₃) which showed shifts at deltaH: 2.48 (2H, f, H-1'), delta 7.75 (1H, brs) 6.02(1H, s, H-6),0.91 (3H, t, H-11'), 1.27 (14H, brs, H-4,/ H- '10'),1.48 (2H, m, H-2'), and1.31(2H, brs, H-3)¹⁰. The advantages and disadvantages of the various processes are considered and also the most easy and convenient methodology is suggested.

In Suthar et al., procedure the Embelia ribes berries were air-dried, small-grained, and stored in a closed container¹¹. 250g of dried powder was taken and extracted with n-hexane using a soxhlet apparatus for 6 h. With cold pet ether, the residue was washed and dissolved in methanol and dichloromethane (DCM) to undergo 24h recrystallization. Once crystallization was complete, the crystals were removed from the mother liquor and washed with n-hexane and DCM to produce crystals of Embelin that were golden in colour¹². Kumara Swamy et al.,also used the soxhlet apparatus for 8 h using methanol as solvent and was concentrated on a rotary evaporator to give reddish brown slurry¹³.

Extraction using n-hexane or alcohol yielded sticky mass by maceration and hot extraction methods, whereas with ether and chloroform extracts crystalline bright product was obtained which showed melting points at 145˚C and 146˚C, respectively. The values are within the limit as specified in the literature.The crystals made with ester had a dull color and had a little higher melting point that is between 150˚C - 160 ̊C. Embelin could not be extracted using the method advised in the Indian Herbal Pharmacopoeia where n-hexane was used in extraction⁶. Similar results were not obtained when Sekar et al., dimethyl ketone extraction method was used⁸.

Embelin was isolated and characterized by forminga crude methanolic extract and E. ribes was then chromatographed using chloroform and methanol as eluent. Thin-layer chromatography checked the presence of a polar fraction of eluted Embelin. Embelin-containing fractions were extracted, concentrated, and yielded 300mg of a crystalline product of a dark orange color. Mass and NMR spectroscopy were used to characterize the isolated component. The molecular ion peak at m/z 294 confirms the mass of the Embelin. In NMR the proton linked to the benzoquinone ring gave off a single proton signal at 6.00ppm and at 7.68ppm the hydroxyl group that is joined to the ring could be seen. At 0.86ppm, a triplet for the three protons of the lengthy alkyl chain's terminal methyl group was present and in the range of 1.25 to 1.29ppm, additional signals for methylene residues of alkyl chains were detected¹⁴.

Many strategies for the isolation of Embelin were reported which had various steps like extractions for a long period, purification, re-crystallization, etc., which resulted in expensive production. Pure Embelin is produced in large quantities via diethyl ether extraction. However, using this solvent carries significant risks for inflammation and other health issues. Additionally, recovering the solvent is not feasible.The use of chloroform for extraction and then washing the dry extract with crude oil ether produced uninflected Embelin with a high yield and efficient purity. As a result, researchers frequently recommend simple cold extraction in chloroform to isolate Embelin¹⁰.

EVALUATION PARAMETERS:

Colour and consistency:

It was anatomized by visual observation. It shows a golden yellow color with crystalline thickness.

Melting point:

A capillary melting point instrument was used to find and confirm the temperature of Embelin. A small quantity of Embelin was taken in the former one-sided sealed capillary and the melting point range was recorded at 143°C - 146°C¹².

TLC identification:

Method 1: 1mg of Embelin was dissolved in 10ml Dichloromethane. 10μl of the test sample was applied on a pre-coated silica gel G plate. The chromatogram was run with a mobile phase consisting of ethyl acetate: toluene: methanol: acid in the ratio of 0.5: 0.4: 0.1: 0.05 in a binary trough chamber. The TLC plate was developed and observed under ultraviolet light, R_f value (R_f 0.752) was compared with the standard Embelin¹⁵.

Method 2: 1g of Vidanga powder was dissolved in 5ml of methanol, sludge, and filtrate for the development of TLC. Solvent system n-propanol: n-butanol: ammonia (1:7:2). Detected by UV at 254nm¹⁶.

Chemical Test:

Embelin was dissolved in pet ether and adulterated with ammonia. The bluish-violet precipitate confirms the presence of Embelin¹².

Content of Embelin by HPLC:

E. ribes fruits (10.0g) were pulverized and homogenized at 200rpm for 20min at room temperature with hexane, ethyl acetate, chloroform, and methanol. Then it was centrifuged for 15min and supernatants were filtered. This was then concentrated to a given volume under reduced pressure at 50°C and the Embelin content was determined in triplicate by HPLC¹⁶. The highest Embelin content and solvent drug ratio were found in ethyl acetate solvent at 45°C for 30min which is 23.71% and 6.25mL/g respectively¹⁷.

ANALYTICAL METHODS:

UV spectrophotometric methods, HPTLC, HPLC, and RP-HPLC are a few of the popular analytical techniques used to assess the potency, purity, and standardization of phytoconstituents The most commonly used analytical instrument for Embelin estimation is HPLC.

Table 2: Analytical methods for the estimation of Embelin

Analytical method	Detector used	Chromatographic conditions	Validation Parameters and Results
TLC¹⁸	UV detector	Chloroform-extracted sample	R_f value: 0.65 Calibration curve linearity range: 5-20µg
HPLC¹⁹	Photodiode array detector	The isocratic phase, which contains 0.1% trifluoroacetic acid in water and methanol (88:12), was used to separate the Embelin at a flow rate of 1 ml/min.	EMB content: 0.44 -33% w/w Linearity range: 5.0-75.0 μg/mL LOD: 20ng LOQ: 50ng
RP-HPLC²⁰	DAD detector	The dried powder was homogenized at 200 rpm for 20 minutes at room temperature with hexane, ethyl acetate, methanol, and chloroform.	LOD: 3.97mg/mL LOQ: 13.2mg/mL Linearity: 15–250mg/mL Precision: 1.43 – 2.87% Recovery: 99.4 – 103.8%
UV Spectrophotometry²¹	UV detector	Dried powder dissolved in methanol	Lambda max: 289nm Linearity: 10-90µg/ml with correlation coefficient: 0.9991 LOD: 3.96µg/mL LOQ: 12µg/mL
HPLC²²	DAD detector	Standard Embelin stock solution: 1 mg/mL in methanol Mobile phase: acetic acid and methanol in a ratio 12:8 Dilutions:15, 25, 50, 100, 150 and 250mg/mL Chromatographic conditions: Pump: LC-8A pump unit Column: reversed-phase Column oven: CTO-10AC VP Rheodyne injector (USA) with volume loop of 20ml	Software: Computerizedchromatography analysis in CLASS-VP version 6.1 Linear range: 15–250mg/mL Regression coefficient (r): 0.9962 Recovery: 99.4–103.8% Intra-day precision: 1.43% Inter-day precision: 2.21%
HPLC¹⁷	UV detector	Chromatographic conditions: Column: C-18 reverse phase Flow rate: 1 mL/min Mobile phase: Methanol and water (88:12v/v) and 0.1% aqueous solution oftrifluoroacetic acid (50:50v/v) in isocratic phase.	Wavelength: 288 nm Embelin content: 32.71%
HPLC²³	UV detector	Chromatographic conditions: Column: C18 reverse phase Mobile phase: trifluoroacetic acid(0.1%) in water and methanol ingradient system Wavelength: 288nm Injection volume: 20μL Flow rate: 1ml/min	Linearity range: 10-30μg/ml Regression coefficient (r): 0.999 LOQ: 0.15μg/ml LOD: 0.051μg/ml Mean retention time: 9.829min Recovery of Embelin: 99 to 100% Robustness studies: %RSD in retention time of change in flow rate: 0.1171 % RSD in retention time of change in pH of 0.1% of TFA in mobile phase: 0.0894
HPTLC²⁴	-	Solvent system: ethyl acetate and chloroform Embelin in dried powder form.	R_f: 0.58 confirmed by LC-MS m/z ratio - 295.1904 retention time - 11.55 min

PHARMACOLOGICAL ACTIVITIES OF EMBELIN:

Analgesic activity:

Embelin have centrally acting non-narcotic and oral analgesic properties. It is more sustainable than morphine as it has higher oral tolerability, a high therapeutic index, a lack of side effects, and an absence of abstinence syndrome¹.

Anthelmintic activity:

Seed oil from Embelia ribes when taken in various dosages like 10mg/ml, 50mg/ml, and 100mg/ml results in the death of Pheretima posthuma worms. The way that worms react to various doses is different i.e. with rise in dose shows a shorter paralysis period. The values are significant in comparison to common piperazine citrate (10mg/ml). Fruit extract from Embelia ribes combined with extract from Veronica anthelmintica seeds provided at 1g/kg and had a significant impact¹.

Antibacterial activity:

Embelia ribes (500mg/50ml) the zone of inhibition is 12 mm in diameter in comparison to nitrofurazone, which has the zone of inhibition of 22 mmagainst Bacillus subtilis. Embelia Ribes had no anti-microbial effects against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Embelin has antibacterial activity against both gram-positive and negative bacteria. Response varies on the dose and bacterial test. Embelin does not possess any antibacterial properties on E. coli and other pathogens at very high levels (100 mg/disc). At (100mg/disc) Embelin had the highest zone of inhibition diameter than standard (kanamycin) at 39 mg/disc against Shigella flexneri, Staphylococcus aureus and sonnei²⁵.

Antifungal activity:

The National Committee for M27-A2 clinical laboratory standard Protocol (NCCLS), a widely used in vitro antifungal susceptibility test, was utilised to evaluate the antifungal activity of Embelia ribes. The NCCLS technique determined that the MIC 50 range for methanolic extract of Embelin against Candida albicans was 120mg/L.

Traumatic brain injury:

One of the most frequent causes of mortality in children is traumatic brain injury. Brain edema and programmed death of neuronal cells are the complications in survivors. Embelin was utilized to determine increased X-chromosomal related apoptosis inhibitor (XIAP) levels that participate in pifithrin-neuroprotective as effects (PFT). Therefore, they strongly suggest that NF-B is involved depending on XIAP regulation to produce the seen neuroprotective effect²⁵.

Hypoxia-ischemia (HI) inducedneurological injury:

When there is a lack of oxygen and blood flow, it is called hypoxia-ischemia (HI), and the result is newborn neurological damage. A study by Hill et al., (2011) examined the caspase-dependent employing Embelin to advance apoptosis, a process known as powerful XIAP inhibitors to demonstrate the influence of gender in several HI-induced cell death pathways²⁶. Embelin binds to BIR3 domain and inhibits XIAP which is X-linked inhibitor of apoptosis protein that increases cell death by caspase dependent route. Compared to HI-induced male rats, female rats have substantial behavioural impairments. These in-vivo results showed that there were substantial variations in male newborns cognitive deficiencies that are more severe than those of female infants who have HI. This phenomenon confirms the data that comparing males' caspase-independent cell death activation than women who trigger the caspase-dependent cascade after newborn ischemia. Embelin is an XIAP inhibitor and concluded that gender affects how cells die after HI injuries and advise that it is important to treat HI, provide a neuroprotection that is sex-specific²⁷.

Anti-inflammatory activity:

Aberrant secretion inhibition decreases edema, mucosal injury, and m-RNA expression of inflammatory cytokines like TNF and IL-6. Due to the down-regulation of inflammatory mediator synthesis and expression. Embelin had the most anti-inflammatory effect in the colon rectal regions²⁸.

Anti-cancer activity:

The anticancer activities of Embelin are mediated by apoptosis, anti-proliferation, anti-angiogenesis and anti-metastasis. Embelin prevents the invasion and migration of prostate cancer cells. It triggers apoptosis that depends on the mitochondria both in vitro and in vivo. Improves the therapeutic effectiveness of radiation therapy in human prostate cancer. Combining EMB and X-ray therapy together improves tumour growth inhibition and apoptosis which results in S-phase cell cycle arrest. EMB promotes apoptosis in dose and time dependent way. The EMB-induced cell death response primarily targets the mitochondria and lysosomes. As a result the mitochondrial membrane is lost²⁸.

MARKET POTENTIAL:

There are several ayurvedic goods on the market with Vidanga as their ingredients. The market for ayurvedic medicines is inadequately regulated due to the acquisition of unauthorised licences and over-the-counter sales which is a serious problem. Due to the fact that this results in adulteration, strict quality criteria such as Embelin markers and laws requiring registration of these formulations, must be put. Few of the available marketed formulations are vidanga churna, vidanga asava and sundar vati.

FUTURE SCOPE:

Embelin being a versatile drug has a lot of potential to be uncovered. Because of historical and cultural factors as well as the perception that they are safer as they come from natural sources, herbal medications have often maintained their popularity. Embelin exhibits all the traits necessary for a drug to pass through the blood-brain barrier (BBB) and have an impact on the central nervous system. Many natural compounds, such as quinone derivatives, are believed to have improved safety and effectiveness profiles and are well-known for their CNS activities. E. ribes fruits, which are rich in hydroxybenzoquinone and are brilliant orange crystalline in colour have gained attention in ethnomedicine. In the traditional systems of medicine, E. ribes fruit has been used to treat problems of the central nervous system (CNS), mental illnesses, and as a brain tonic. The results of these investigations reveal that Embelin could be a potent neuroprotective drug and adjuvant therapy for cerebral stroke.In addition, a lot of people in wealthy nations have started using complementary or alternative medicines, including herbal remedies.Embelin is safe when administered orally to rodents up to 3g/kg after acute exposure. Embelin can be utilised as a possible "lead compound" to create other "safer" compounds and create newer carbapenem adjuvants to fight bacterial strains that produce NDM-1. According to studies, Embelin may one day be used to treat epilepsy and memory problems. The World Health Organisation (WHO) claims that the use of traditional medicines has gained international attention. For their main healthcare needs, a sizable segment of society in many developing nations heavily relies on traditional healers and phytomedicine.

CONFLICT OF INTEREST:

In relation to this inquiry, the authors have no conflicts of interest.

ACKNOWLEDGEMENT:

Authors are thankful to Bharati Vidyapeeth’s College of Pharmacy, Sector-8, C.B.D., Belapur, Navi Mumbai for providing the necessary facilities.

REFERENCE:

1. Lal B. Mishra N. Importance of embelia ribes: an update. International Journal of Pharmaceutical Science and Research. 2013; 4(10): 3828-38. http://dx.doi.org/10.13040/IJPSR.0975-8232.4(10).3823-38.

2. Sudhakaran V. Botanical pharmacognosy of the fruit of Embelia ribes Burm. F. Pharmacognosy and Natural Products. 2015; 1(1): 103.10.4172/2472-0992.1000103.

3. Xavier E. Kani J. Bioactivity, analytical techniques and formative approaches for Embelin. Research Journal of Pharmacy and Technology. 2022; 14(1): 487-92. 10.5958/0974-360X.2021.00089.5.

4. Narayanaswamy R. Gnanamani A. 5-dihydroxy-3-undecyl-1, 4-benzoquinone (embelin)-a second solid gold of India- a review. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(2): 23-30.

5. Karpakal S. Rajasekharan PE. Ethnopharmacological uses of Embelia ribes Burm. F. – A review. Journal of Pharmacy and Biological Sciences. 2014; 9(3): 23-30

6. Indian Herbal Pharmacopoeia. Revised new edition. Mumbai; Indian Drug Manufacturers Association, 2002; 206-213.

7. Bisrat D. Mazumder A. Asres K. In vitro antimicrobial activity of a semi-synthetic derivative of embelin. Ethiopian Pharmaceutical Journal. 2014; 30(1): 50-6.http://dx.doi.org/10.4314/epj.v30i1.5.

8. Mahendran S. Badami S. Subban R. Thippeswamy BS. Veerapur VP. Synthesis and evaluation of analgesic and anti-inflammatory activities of most active free radical scavenging derivatives of embelin -A structure-activity relationship. Chemical and Pharmaceutical Bulletin. 2011; 59(8): 913-919. 10.1248/cpb.59.913.

9. Latha C. Microwave-assisted extraction of embelin from Embelia ribes. Biotechnology Letters. 2007; 29(2): 319-21. 10.1007/s10529-006-9243-z.

10. Pundarikakshudu K. Joshi H. Shah P. Panchal S. A simple, facile method for isolation of embelin from fruits of embelia ribes Burm.F. (Vidang). Indian drugs. 2016; 53(2): 23-27.

11. Suthar M. Patel R. Hapani K. Patel A. Screening of Embelia ribes for antifungal activity. International Journal of Pharmaceutical Sciences and Drug Research. 2009; 1(3): 203-206.

12. Kaur V. Hallan SS. Kalia N. Mishra N. Isolation of embelin from and evaluation of its anti-cancer potential in Embelia ribes breast cancer. Asian Journal of Pharmacy and Pharmacology. 2015; 1(1): 34-35

13. Kumara Swamy HN. Krishna V. Shankarmurthy K. Abdul Rahman B. Mankani KL. Mahadevan KM et al. Wound healing activity of embelin isolated from the ethanol extract of leaves of Embelia ribes Burm. Journal Ethnopharmacology. 2007; 109(3): 529-534. 10.1016/j.jep.2006.09.003.

14. Sidana A. Dhindsa NK. Farooq U. Isolation, biotransformation and evaluation of antibacterial activity of embelin from Embelia ribes. Journal of Phytochemistry and Ayurvedic Heights. 2012; 2(13): 6-7.

15. Chauhan S. Singh B. Agarwal S. A TLC identification and spectrophotometric estimation of Embelin in Embelia ribes. Ancient Science of Life. 1999; 19(13): 46-48.

16. Laddha KS. Extraction and isolation of Phytochemicals. A practical approach. Published by yucca enterprises, Mumbai. First edition: 2019; 25-27.

17. Alam MS. Damanhouri ZA. Ahmad A. Abidin L. Amir M. Aqil M. Khan SA. Mujeeb M. Development of response surface methodology for optimization of extraction parameters and quantitative estimation of embelin from Embelia ribes Burm by high Performance Liquid Chromatography. 2015; 11(42): 166-172. 10.4103/0973-1296.157722.

18. Chauhan SK. Singh BP Agarwal S. A TLC identification and spectrophotometric estimation of embelin in Embelia ribes. Ancient Science of Life. 1999; 19(1-2): 46.

19. Shelar R. Maurya C. Tekale P. Katkar K. Naik V. Suthar A. Chauhan VS. Embelin-An HPLC method for quantitative estimation in Embelia ribes Burm. F. International Journal of Pharmaceutical and Clinical Research. 2009; 1(3):146-9.

20. Madhavan SN. Arimboor R. Arumughan C. RP‐HPLC‐DAD method for the estimation of embelin as marker in Embelia ribes and its polyherbal formulations. Biomedical Chromatography. 2011; 25(5): 600-5. 10.1002/bmc.1489.

21. Prasad SB. Bist M. Verma SP. Development and validation of the UV-spectrophotometric method for determination of embelin. International Journal of Pharmaceutical Quality Assurance. 2016; 7(3): 51- 54.

22. Madhavan SN. Arimboor R. Arumughan C. RP HPLC DAD method for estimation of embelin as marker in Embelia ribes and its polyherbal formulations. Biomedical Chromatography. 2010; 25(5): 600-605. 10.1002/bmc.1489.

23. Gondaliya AV. Vikani K. Kapupara PP. Development and validation of analytical method for simultaneous estimation of berberine hydrochloride and embelin in polyherbal formulation. International Bulletin of Drug Research. 2014; 4(6): 35-44.

24. Swami D. Fulzele D. Malpathak N. Identification and quantification of Embelin by validated HPTLC method and confirmation by LC-MS from mangrove plant aegiceras corniculatum L. Journal of Chemical and Pharmaceutical Research. 2017; 9(1): 168-73.

25. Lum P. Othman S. Molecules of interest – Embelin – a review. Research Journal of Pharmacy and Technology. 2020; 13(7): 3485-93.10.5958/0974-360X.2020.00618.6.

26. Koppal A. Vagdevi H. Therapeutic effect of Embelin and Levodopa combination in rotenone induced parkinson’s disease in mice on neurobehavioural changes. Research Journal of Pharmacy and Technology. 2023; 16(5): 2107-4.

27. Kundap UP. Bhuvanendran S. Kumari Y. Othman I. Shaikh Mohd F. Plant derived by phytocompound, Embelin in CNS disorders: a systematic review. Frontiers in Pharmacology. 2017; 8.

28. Othman S. Mahendran S. In-vitro antioxidant and cytotoxic activities of silver nanoparticles of Embelin isolated from Embelia ribes. Research Journal of Pharmacy and Technology. 2019; 12(9): 4080-84.

Received on 24.07.2023 Modified on 21.11.2023

Research J. Pharm. and Tech 2024; 17(8):4083-4088.

DOI: 10.52711/0974-360X.2024.00633