Bioactive (+)-Catechin-3ꞌ-O-rhamnopyranoside from Neocarya macrophylla (Sabine) Prance (Chrysobalanaceae)

ABSTRACT

In this study, a flavan-3-ol glycoside, (+)-catechin-3ꞌ-O-rhamnopyranoside isolated from the stem bark extract of N. macrophylla was evaluated for its antivenom, analgesic, anti-inflammatory, and antimicrobial activities. The compound (at 1, 5 and 10 mg/kg) was able to protect mice against lethality induced by Naja nigricollis venom with 100%, 40%, and 20% survival rate, respectively; it was also found to be 33.7% effective in combating the hydrolytic actions of N. nigricollis PLA₂ enzyme; the compound (at 2.5 and 10 mg/kg) exhibited significant (p < 0.05) analgesic effect in the formalin test which was higher than that observed by the standard drug, piroxicam (10 mg/kg) and there was 45.0% and 41.2% inhibition of inflammation by the compound at the 3rd & 4th hours. In addition, the compound was found to be effective against S. aureus, VRE, S. feacalis, E. coli, P. aeruginosa, P. vulgaris, C. albicans, and C. krusei with mean zone of inhibition ranged between 28 and 36 mm and an MIC and MBC/MFC values ranging from 6.25 to 25.0 µg/mL and 12.5–50.0 µg/mL, respectively. Thus, bioactivities of (+)-catechin-3ꞌ-O-rhamnopyranoside were reported for the first time.

KEYWORDS:

• Neocarya macrophylla
• (+)-catechin-3ʹ-O-rhamnopyranoside
• antivenom
• analgesic
• anti-inflammatory
• antimicrobial

Introduction

Snakebite is a global medical problem especially in the rural areas of the tropics with about 5.4 million bites each year resulting in 1.8 to 2.7 million cases of envenomings, accompanied by 81,1400 to 137,880 deaths and three times as many amputations and other permanent disabilities each year [1,71]. The highest burden of snakebites is in Asia and Sub-Saharan Africa; studies have indicated that, over 314,000 bites, 7300 deaths, and nearly 6000 amputations occur from snakebites annually in Sub-Saharan Africa and Nigeria, by poisonous snakes from two different families of elapids and viperids [2–4]; but the precise incidence is grossly under-estimated due to lack of data and low access to community health facilities [4-6]. Bites by venomous snakes may lead to complications such as, tissue necrosis, severe pain, swelling, bruising, abdominal pain, diarrhea, blindness resulting from spitting cobra, fetal loss, edema, wound infection, tetanus, severe paralysis that may prevent breathing, bleeding disorders that can lead to fatal hemorrhage, irreversible kidney failure and severe local tissue destruction that can cause permanent disability and limb amputation [1,7–10]. However, mortality rate has been reported to be higher in children due to larger amount of venom injected per body weight [11–14]. Due to the wide spectrum of clinical symptoms, therapy using multiple drugs such as anticholinesterase, antibiotics, and analgesics is required [15]. The currently used Antisnake venoms are accompanied by severe side effects and are largely unavailable due to access and cost [4]. These drawbacks necessitate the search for newer agents with improved therapeutic effects.

Most communities in the sub-Saharan Africa, with poor access to health services resort to alternative therapy, which involve the use of extracts from medicinal plants as antivenoms [16]. Several medicinal plants have been screened for their antivenom property [16–17] (Walter et al., 2000; Gomes et al., 2010), but researches mostly end up at preliminary stage of biological testing of the crude extracts leaving behind the crucial stages of isolation and characterization of bioactive constituents responsible for the observed effect.

Neocarya macrophylla (Sabine) Prance (Chrysobalanaceae) is widely distributed along coastal savannahs from Senegal to Liberia, woody savannahs of Southern Mali, Niger and Northern Nigeria [18,19]. It has been used extensively in the Northern part of Nigeria in ethno-medicine to treat numerous diseases which include; asthma, skin infections, treatment of wounds, dysentery, inflammations, pain, snake bite, diarrhea, pulmonary troubles, ear and eye infections, among others [20,21]. Chemical investigations on the stem bark of the plant were limited to steroids such as stigmasterol and flavanols; dimer of flavan-3-ol glycoside i.e. Bis-(5,7-diacetyl-catechin-4’-α-rhamnopyranoside) has hitherto been isolated [22]. GC-MS analysis of the chloroform leaf extract of N. macrophylla revealed the presence of flavonoids, steroids, palmitoleic acid, tannins, glycosides, alpha and beta tacopherol [25]. Pharmacological actions of the stem bark of N. macrophylla has been confined to analgesic [23], antimicrobial [24] and antivenom [25] activities; Isaka et al. [25] has reported the antimicrobial activity of the ethanol leaf extract of the plant; the extracts and fractions of N. macrophylla leaf exhibited remarkable antibacterial and antibiofilm activities against S. aureus and P. aeruginosa [26]. Antimicrobial activity of stigmasterol from the stem bark of the plant has also been reported [27]. The effect of the saponin-rich fractions of the stem and root bark of the plant on murine models of pain and inflammation has been reported [28]. This is a follow-up work to isolate bioactive compound(s) from the stem bark of the plant. We now report the isolation and characterization of (+)-Catechin-3ꞌ-O-rhamnopyranoside from the stem bark of Neocarya macrophylla and its bioactivities.

Materials and methods

General procedures

NMR data were recorded on a Bruker AVANCE spectrometer (600 MHz) with residual solvent as an internal standard. Melting point was determined on an Electrothermal melting point apparatus. TLC was carried out using silica gel 60 GF₂₅₄ pre-coated aluminum sheets manufactured by Sigma Aldrich, Germany. Column chromatography was conducted using LOBA Cheme silica gel (60–200) mesh while gel filtration chromatography was performed using sephadex LH-20 (Sima, Spruce street, St. Louis, MO, USA). Spots on TLC plates were visualized by spraying with 10% H₂SO₄ followed by heating at 105°C for 10 min.

Plant material

The plant sample of N. macrophylla was collected in October 2015 at Jega Local Government Area of Kebbi State. It was identified by Namadi Sanusi at the Herbarium section, Department of Biological Sciences, Ahmadu Bello University by comparing with herbarium reference voucher specimen (No. 3197). The stem bark was shade-dried, pulverized to powder, labeled and stored at room temperature for use.

Extraction and isolation

The dried powder (3000 g) was extracted with 90% methanol using maceration method for 6 days. After evaporation of the solvent with the aid of rotary evaporator at 40°C, the extract was suspended in distilled water, filtered and successively partitioned with n-hexane (1 L), dichloromethane (1 L), ethylacetate (2.5 L) and n-butanol (2.5 L) to obtain hexane, dichloromethane, ethylacetate, n-butanol and the residual aqueous fractions. The ethylacetate fraction (13.0 g) was gradiently eluted in a silica gel-packed column (5 × 75 cm) using different solvent combinations starting with n-hexane: ethylacetate (1:1), ethylacetate (100%) to ethylacetate: methanol (8:2). Thirty (30) mL each of a total of 426 fractions was collected and combined based on their TLC profile to give (12) major fractions coded EA-EL. Repeated gel filtration of fraction EH with sephadex LH-20 using methanol as eluting solvent gave three major fractions EH1–EH3. Fraction EH1 further purified using gel filtration repeatedly yielded Compound 1 (82.5 mg) and it was subjected to Ferric chloride, Fehling’s tests and spectroscopic analysis to elucidate its structure.

Pharmacological studies

Experimental animals

Swiss albino mice and adult Wister rats of either sex weighing (18–22 g) and (170–200 g), respectively, were obtained from the Animal House Facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria. They were fed with laboratory diet and water ad libitum and maintained under standard conditions (12 h light and 12 h dark cycle) in propylene cages at room temperature. All experimental procedures were approved by the Animal Right and Ethics Community of the University.

Venom sample

The venom of an adult Naja nigricollis was collected by the milking method of Markfalane [29]. It was lyophilized and stored at 4°C until required.

Standard drugs

Piroxicam (Pfizer, Pakistan). Lyophilized polyvalent snake venom antiserum by VINS Nioproduct Limited, Andra Pradesh, India. Batch. No: 01AS14054, MD: July 2015, ED: June 2018.

Microbial organisms

Clinical isolates were obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria and comprised of Staphylococcus aureus, Vancomycin-Resistant Enterococci, Streptococcus pyogenes, Streptococcus faecalis, Escherichia coli, Corynebacterium ulcerans, Streptococcus pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris, Candida albicans Candida tropicalis Candida krusei. All the isolates were checked for purity and maintained in slants of nutrient agar (for bacteria) and in slants of sabouraud dextrose agar for fungi

Antisnake venom studies

Lethality assay of the venom

The LD₉₉ of the venom was determined using the method described by [30]; the N. nigricollis venom was reconstituted with normal saline and concentrations ranging from 0.1 to 0.6 mg/mL were obtained. Thirty-five (35) mice were divided into seven (7) groups of five (5) mice each and were injected i.p. with different doses of the reconstituted venom. The control group received only normal saline (0.2 mL each, i.p.). The LD₉₉ was calculated by probit analysis [31] of death occurring within 24 h of venom injection.

Venom detoxification by the compound

The method described by [32] was employed in this study. Twenty (20) mice were divided into four groups (n = 5). Group 1 (control) received the MLD (LD₉₉, 5.75 mg/kg) of N. nigricollis venom only. Groups 2, 3 and 4 (treatment groups) received an equivalent of the MLD of the venom containing 1, 5 and 10mg/kg of compound 1, previously incubated at 37°C for 10 min and the animals were observed for mortality for 24 h. The route of administration was intraperitoneal.

Phospholipase A₂ enzyme assay

Acidimetric method, as reported by Tan and Tan [33], was used to evaluate the PLA₂ enzyme inhibition potential of compound 1.

Analgesic studies – formalin test in rats

Fifteen rats were divided into five groups of three rats and were administered with either normal saline (1 mL/kg, i.p.), compound 1 (2.5, 5.0 and 10.0 mg/kg, i.p.) or piroxicam (10 mg/kg, i.p.). Thirty minutes after this treatment, 50 µL of a freshly prepared 2.5% solution of formalin was injected subcutaneously under the plantar surface of the left hind paw of each rat. The rats were placed individually in an observation chamber and monitored for 1 h. The severity of pain response was recorded for each rat based on the following numerical scale: (0) rat walked or stood firmly on the injected paw; (1) the injected paw was favored or partially elevated; (2) the injected paw was clearly lifted off the floor; (3) the rat licked, chewed or shook the injected paw. Anti-nociceptive effect was determined in two phases. The early phase (phase 1) was recorded during the first 5 min, while the late phase (phase 2) was recorded during the last 45 min with a 10 min lag period in-between both phases [34].

Anti-inflammatory studies – formalin-induced inflammation in rats

Formaldehyde 2.5% v/v was used as inflammogen [35]. Rats were divided into five groups of three rats each. Thirty minutes before injection of 2.5% v/v formalin (50 µL volumes in the subplantar region of the left hind paw of the rat), the groups were treated i.p. as follows:

Group I: received (1 mL normal saline per kg, as negative control).

Groups II, III and IV: received (compound 1, at 2.5, 5.0 and 10.0 mg per kg, respectively).

Group V: received (10 mg piroxicam per kg, as positive control). The paw diameter (cm) was measured using vernier caliper at an interval of 1 h for 4 h.

Antimicrobial screening

Compound 1 was tested for antimicrobial activity against clinical isolates of S. aureus, VRE, S. pyogenes, S. feacalis, E. coli, C. ulcerans, S. pneumonia, P. aeruginosa, P. vulgaris, C. albicans, C. tropicalis and C. krusei, as previously described by Vollekova et al. [36]. Mueller Hinton agar was used as the growth medium for the microbes. The medium was prepared according to the manufacturer’s instructions and sterilized at 121°C for 15 min. Nutrient broth and Sabdroud dextrose broth were used for antibacterial and antifungal evaluations, respectively. It was poured into sterile petri dishes, allowed to cool and solidify. The sterilized medium was seeded with 0.1 mL of standard inoculum of the test microbe; the inoculum was spread evenly over the surface of the medium by the use of a sterile swab. Standard sterile cork borer of 6 mm in diameter was used to bore a well at the center of each inoculated medium. The wells were filled with 0.1 mL of the solution of the compound and allowed to diffuse for 1 h. Incubation of the inoculated medium was made overnight at 37°C and 25°C for bacteria and fungi, respectively, after which the medium was observed for the zone of inhibition of growth; the tests were done in duplicates and the zone of inhibition was measured with a transparent ruler. The mean of the results was recorded in millimeters (mm). The MICs of the compound were determined using the broth dilution method. Two-fold serial dilutions of the compound in the sterile broth were made to obtain the concentrations of 100, 50, 25, 12.5, 6.25 and 3.13 µg/mL; 0.1 mL suspension of the standard inoculum of the test microbe was then inoculated into the different concentrations of the compound. The tubes were incubated at 37°C for 24 h and 25°C for 48 h for bacteria and fungi, respectively, after which the plates were observed for turbidity (growth). The MIC was defined as the lowest concentration of the compound inhibiting the visible growth of each micro-organism. MBC/MFC was carried out to determine whether the test microbes were killed or only their growth was inhibited. Mueller Hinton agar broth was prepared, sterilized at 121°C for 15 min, and transferred into sterile petri dishes to cool and solidify. The contents of the MIC in the serial dilution were sub-cultured into the prepared medium and incubated at 37°C for 24 h; the plates were observed for colony growth; the MBC/MFC was the plate with lowest concentration of the compound in serial dilution without colony growth [36].

Statistical analysis

The data were analyzed using Chi-Square and One-Way ANOVA followed by post hoc test using the SPSS software (version 22). The results were expressed as Mean ±SEM. Values of p < 0.05 were considered significant.

Results

Characterization of compound 1

(+)-Catechin-3ꞌ-O-rhamnopyranoside. Dark brown solid substance, m.p. 206–208°C; The ¹H-NMR spectrum (CD₃OD) of compound 1 revealed the following chemical shift values/integration: δ_H 5.91 (1H, d, J = 1.9 Hz, H-6), 5.97 (s, H-8), 7.14 (1H, d, J = 8.3 Hz, H-5ꞌ),6.95 (1H, d, J = 2.0 Hz, H-2ꞌ),6.86 (1H, dd, J = 2.0, 8.3 Hz, H-6ꞌ), 4.65 (1H, d, J = 7.5 Hz, H-2),4.03 (m, H-3), 2.89 (IH, dd, J = 5.4, 16.1 Hz, H-4b), 2.57 (1H, dd, J = 8.2, 16.1 Hz, H-4a), 1.23 (J = 6.0 Hz, H-6ꞌꞌ), 4.15 (1H, dd, 1.3, 2.1 Hz, H-2ꞌꞌ), 5.42 (1H, d, J = 1.3, H-1ꞌꞌ), 3.98 (1H, J = 3.3, 9.5 Hz, H-6ꞌꞌ). ¹³C-NMR (600 MHz, CD₃OD); δ_C 156.1 (C-9), 155.4 (C-5), 156.4 (C-7), 99.8 (C-10), 134.4 (C-1ꞌ), 147.2 (C-4ꞌ) and 144.1 (C-3ꞌ), 81.2 (C-2), 67.4 (C-3), 27.1 (C-4), 94.0 (C-6), 95.0 (C-8), 118.5 (C-6ꞌ), 117.1 (C-5ꞌ), 114.8 (C-2ꞌ), 100.0 (C-1ꞌꞌ), 70.6 (C-2ꞌꞌ), 70.8 (C-3ꞌꞌ), 72.6 (C-4ꞌꞌ), 69.4 (C-5ꞌꞌ), 16.6 (C-6ꞌꞌ).

Antisnake venom studies

The minimum lethal dose (LD₉₉) of the venom estimated by probit was 5.75 mg/kg. Compound 1 has demonstrated significant (p < 0.05) antivenom activity against N. nigricollis venom in mice. Maximum protection (100%) against venom-induced lethality was observed at the lowest dose (1 mg/kg) compared to the control group; 40% and 20% survival rate was recorded at 5 and 10 mg/kg of compound 1, respectively (Table 3).

Table 1. 1D and 2D NMR spectral data summary for compound 1 (CD₃OD).

Position	δ ^1H	δ ^13C	DEPT	COSY	HMBC
1	-	-	-	-	-
2	4.65	81.2	CH	H-3	C9, C1ʹ, C6ʹ, C2ʹ, C2
3	4.03	67.4	CH	H-4a, H-4b	-
4	2.89	27.1	CH2	H-4a	C7, C10, C2
	2.57		CH2	H-4b	C7, C10, C2
5	-	155.4	C	-	-
6	5.91	95	CH	-	-
7	-	156.4	C	-	-
8	5.97	94	CH	-	C9, C10
9	-	156.1	C	-	-
10	-	99.6	C	-	-
1’	-	134.4	C	-	-
2’	6.95	114.8	CH	-	C2, C6ʹ, C3ʹ, C4
3’	-	144.4	C	-	-
4’	-	147.2	C	-	-
5’	7.14	117.1	CH	H-6’	C4ʹ, C3ʹ, C1’
6’	6.86	118.5	CH	H-8’	C2, C2ʹ, C3’
Rhamnose
1ʹ’	5.42	100	CH	H3ʹ, H-2”	-
2ʹ’	4.15	70.6	CH	H-1”	-
3”	3.98	70.8	CH	H-4”	C4”
4”	3.51	72.6	CH	-	C2”, C6”
5”	3.84	69.4	CH	H-4”	-
-CH3	1.23	16.6	CH3	-	-

Table 2. ¹H and ¹³C-NMR data of compound 1 compared with reported literature.

Position	δ ^13C	δ ^1H (J in Hz)	δ ^13C [46]	δ ^1H (J in Hz) [46]
1	-	-	-	-
2	81.2	4.65 (d, 7.5)	83.1	4.62 (d, 8.0)
3	67.4	4.03 (m)	76.1	3.93 (m)
4	27.1	2.89 (dd, 5.4, 16.1)	28.1	2.88 (dd, 5.5, 16.0)
		2.57 (dd, 8.2, 16.2)		2.64 (dd, 8.5, 16.0)
5	155.4	-	157.1	-
6	94	5.91 (d, 1.9)	96.5	5.94 (d, 2.3)
7	156.4	-	157	-
8	95	5.97 (s)	95.6	5.86 (d, 2.3)
9	156.1	-	158.1	-
10	99.6	-	100.8	-
1’	134.4	-	132.1	-
2’	114.8	6.95 (d, 2.0)	115.2	6.84 (d, 1.8)
3’	144.1	-	146.4	-
4’	147.2	-	146.5	-
5’	117.1	7.14 (d, 8.3)	116.2	6.77 (d, 8.0)
6’	118.5	6.86 (dd, 2.0, 8.4)	120	6.72 (dd, 1.8, 8.0)
Rhamnose
1”	100	5.42 (d, 1.3)	102.3	4.29 (d, 1.4)
2”	70.6	4.15 (dd, 1.3, 2.1)	72.1	3.51 (dd, 1.8, 3.2)
3”	70.8	3.98 (dd, 3.3, 9.5)	72.4	3.57 (dd, 3.2, 9.6)
4”	72.6	3.51 (t)	74.1	3.31 (m)
5”	69.4	3.84 (m)	70.5	3.68 (m)
-CH3	16.6	1.23 (d, 6.0)	18.1	1.25 [d, 6.2)
Compound 1 = CD3OD; 600 MHz			[46] = CD3OD; 400 MHz

Table 3. In vitro detoxifying effect of compound 1 against N. nigricollis venom.

Group	Treatment (mg/kg)	% Survival (within 24 h)
1	LD99	-
2	LD99 + 1	100*
3	LD99 + 5	40*
4	LD99 + 10	20*

*% survival of mice was significantly higher than control group using Chi-Square at p < 0.05; n = 5.

The compound was able to inhibit the action of N. nigricollis PLA₂ enzyme activity with 33.7% inhibition which was lower compared to the standard ASV (72.9%) (Figure 2).

Figure 1. Structure of compound 1.

Analgesic studies

The result of the analgesic activity of compound 1 is shown in Table 4. Compound 1 exhibited significant (p < 0.05) analgesic effect in both phases; the compound at the highest (10 mg/kg) and medium (5 mg/kg) doses was able to inhibit the pain response after injection of formalin in the first phase with mean pain scores of 2.33 ± 1.2 and 2.67 ± 2.1, respectively, which was better than the effect (4.67 ± 2.7) observed for the standard drug, piroxicam at 10 mg/kg (Table 4).

Table 4. Effect of compound 1 on formalin-induced pain in rats.

Shift Mean Pain Score here
Treatment (mg/kg)	First phase	Second phase
Normal saline	12.33 ± 2.3	32.00 ± 0.6
Compound 1(2.5)	6.00 ± 1.0	16.67 ± 2.3
Compound 1(5.0)	2.67 ± 2.1*	22.33 ± 2.2
Compound 1 (10.0)	2.33 ± 1.2	20.67 ± 1.8
Piroxicam (10.0)	4.67 ± 2.7*	16.67 ± 2.3*

Each value represents mean ± SEM. *p < 0.05 compared with normal saline (One-way ANOVA); n = 3.

Anti-inflammatory studies

Compound 1 and the standard drug, piroxicam reduced the increase in paw size induced by formalin at all the tested doses (2.5, 5.0 and 10 mg/kg) when compared to the negative control (Table 5). Highest inhibition of inflammation was observed at the highest dose (10 mg/kg); however, the lowest dose (2.5 mg/kg) had 45.0% and 41.2% inhibition of inflammation at the 3rd and 4th hours, respectively. The standard drug, piroxicam at 10 mg/kg had 65% inhibition of inflammation (Table 5).

Table 5. Effect of compound 1 on formaldehyde induced inflammation in rats.

shift % inhibition of inflammation
Treatment (mg/kg)	1h	2 h	3 h	4 h
N/saline	-	-	-	-
Compound 1 (2.5)	18.8	16.7	45.0	41.6
Compound 1 (5.0)	6.3	5.5	-	5.9
Compound 1 (10.0)	43.8	38.9	45.0	41.2
Piroxicam (10.0)	50.0	50.0	65.0	41.2

Each value represents mean ± SEM. *p < 0.05 compared with normal saline (One-way ANOVA); n = 3.

Antimicrobial studies

The result of antimicrobial activity of compound 1 is shown in Table 6. Twelve microorganisms (including nine bacteria and three fungi) were screened. The compound was active against all the test organisms except S. pyogenes, C. ulcerans, S. pneumoniae and C. feacalis with zone of inhibition ranged between 28 and 36 mm; the most sensitive organism was P. aeruginosa (36 mm) while the least sensitive was the fungi, C. albicans (28 mm); the zones of inhibitions were slightly lower than those observed for the standard drugs (ciprofloxacin and fluconazole). The compound exhibited higher inhibition zone against C. krusei (35 mm) compared to the standard antifungal agent (fluconazole) with mean zone of inhibition of 32 mm. The MIC values ranged from 6.25 to 25.0 µg/mL while MBC/MFC ranged from 12.5 to 50 µg/mL.

Table 6. Antimicrobial activity of compound 1 (100 µg/mL).

Test organisms	Compound 1	Ciproflaxin (5 µg/mL)	Fluconazole (5 µg/mL)
S. aureus	32 (25.0/50.0)	38	NT
VRE	31 (6.25/25.0)	32	NT
S. pyogenes	-	35	NT
S. feacalis	30 (25.0/50.0)	34	NT
E. coli	29 (12.5/25.0)	30	NT
C. ulcerans	-	30	NT
S. pneumoniae	-	32	NT
P. aeruginosa	36 (25.0/50.0)	-	NT
P. vulgaris	30 (12.5/25.0)	35	NT
C. albicans	28 (6.25/12.5)	NT	35
C. tropicalis	-	NT	32
C. krusei	35 (12.5/25.0)	NT	32

Key: Values outside the brackets indicate zones of inhibition (mm). The first value in parenthesis indicates MIC while the second value indicates MBC/MFC for bacteria and fungi as the case maybe. – = no activity, NT = not tested.

Discussion

The study was inspired by the widespread ethnomedicinal use of the plant N. macrophylla in the management of snakebite, microbial infections, pain, and anti-inflammatory diseases. Preliminary results provided promising results and validated the ethnomedicinal claims [25] . Inspired by these results, the ethylacetate soluble fraction of the methanol stem bark extract which appeared to be promising was subjected to chromatographic studies in order to isolate and evaluate bioactive compound(s) responsible for the observed antivenom, analgesic, anti-inflammatory, and antimicrobial effects. Thus, the study led to the isolation of a flavonoid. Compound 1 was isolated as a dark brown solid substance. It reacted positively to ferric chloride reagent suggesting the presence of phenolic nucleus and the presence of sugar moiety was confirmed via Fehling’s test [37]. The basic skeletal structure of compound 1 was confirmed from the ¹H-NMR data; the presence of two meta-coupled protons (ring A) at δ_H 5.91 (1H, d, J = 1.9 Hz) and 5.97 (brs) corresponding to H-6 and H-8, respectively, was observed from the calculation of J values. An ABC ring system was clearly discerned from the proton signals at δ_H 7.14 (1H, d, J = 8.3 Hz, H-5ꞌ), δ_H 6.95 (1H, d, J = 2.0 Hz, H-2ꞌ) and δ_H 6.86 (1H, dd, J = 2.0, 8.3 Hz, H-6ꞌ) and the protons at δ_H 4.65 (1H, d, J = 7.5 Hz, H-2) and δ_H 4.03 (m, H-3) were due to the presence of an oxygenated aliphatic moiety and the 2, 3-trans stereochemistry was depicted by the high coupling constant value (J_H-2-H-3 = 7.5 Hz) of the heterocyclic ring C which confirmed compound 1 to be a catechin rather than an epicatechin [38–41]. Two methylene proton signals at δ_H 2.89 (IH, dd, J = 5.4, 16.1 Hz) and δ_H 2.55 (1H, dd, J = 8.2, 16.1 Hz) representing H-4b and H-4a, respectively, further confirmed the presence of an AX-type ring C typical of 3-flavan-type flavonoids [35,36]. The sugar moiety was defined by the resonances at δ_H 3.51–5.42 with integral intensities of five protons and a doublet at δ_H 1.23 (J = 6.0 Hz) typical of the – CH₃ group which suggests the sugar to be a rhamnoside [42–43]. The anomeric configuration was assigned based on the coupling constant (J = 1.3 Hz) of the anomeric proton at δ_H 5.42. Proton signal at δ_H 4.15 (1H, dd, 1.3, 2.1 Hz) was assigned to H-2” and H-3” appeared as a dd at δ_H 3.98 (1H, J = 3.3, 9.5 Hz). H-4” resonated as a triplet (δ_H 3.51) with coupling constant J = 9.5. Protons were observed with J values (J = 3.4 Hz) all across at δ_H 3.84 and was assigned to H-5” of the sugar.

The ¹³C-NMR (600 MHz, CD₃OD) and DEPT experiments of compound 1 indicated the presence of seven aromatic carbon peaks. The ¹³C-NMR spectrum showed a resonance at δ_C 81.2 which appeared in a lower field further suggesting the compound to be a catechin [38–41]. The chemical shift values at δ_C 100.0, 70.6, 70.8, 72.6 and 69.4 were characteristic of sugar absorptions and the – CH₃ absorption at δ_C 16.6 suggests the sugar to be a rhamnose which is in consistency with the ¹H-NMR data [44,45]. All protonated carbons were assigned by the HSQC spectra (Table 1); the 2D ¹H-¹H COSY correlation spectrum of compound 1 revealed the protons that are adjacent to one another. Major correlations observed are; δ_H 7.14 (H-5ʹ) and 6.86 (H-6ʹ) which confirmed the assignment on ring B; Correlations at δ_H 4.65 (H-2) and 4.03 (H-3), δ_H4.03 (H-3) and 2.89, 2.55 (H-4b, a), δ_H 2.89 (H-4b) and 2.55 (H-4a) confirmed the presence of ring C, typical of flavan-3-ols. The anomeric proton of the sugar moiety δ_H 5.42 (H-1”) correlated with the proton at δ_H 4.15 (H-2”). Other correlations observed within the sugar moiety include; δ_H 3.98 (H-3”) and 3.51 (H-4”), δ_H 3.84 (H-5”) and 3.51 (H-5”). The HMBC between the protons and carbons was used to establish the connectivity between the sub-structures in the molecule. Long-range correlation was observed at δ_H7.14 and δ_C 147.2 (C-4ʹ), 144.1 (C-3ʹ) and 134.4 (C-1ʹ); δ_H 6.95 and δ_C 81.2 (C-2), 118.5 (C-6ʹ), 144.1 (C-3ʹ) and 147.2 (C-4ʹ); δ_H 6.86 and δ_C 81.2 (C-2), 114.8 (C2ʹ), 144.1 (C-3ʹ) which confirmed the presence of ring B system. The presence of an ABC-system was ascertained via the correlations observed at δ_H 5.97 and δ_C 156.4 (C-9) and 99.8 (C-10); δ_H 4.65 and δ_C 156.4 (C-9), 134.4 (C-1”), 118.5 (C-6ʹ), 114.8 (C-2ʹ), 67.4 (C-2) and 27.1 (C-4); δ_H 2.89, 2.55 (H-4b, a) and δ_C 155.4 (C-7), 99.8 (C-10), 81.2 (C-2) and 67.4 (C-4). The anomeric proton signal at δ_H 5.42 (H-1”) exhibited a long-range correlation with δ_C 144.1 (C-3ʹ) and 70.8 (C-3”) confirming the linkage of the aglycone to the rhamnosyl chain at C-3. Other HMBC correlations for the sugar moiety are δ_H 3.98 and δ_C 72.6 (C-4”); δ_H 3.51 and δ_C 70.6 (C-2”) and 16.6 (C-6”) (Table 1). Based on the 1D and 2D-NMR spectral data (Table 1) and comparison with existing data in the literature [46, Table 2], the structure of compound 1 (Figure 2) was confirmed as (+)-Catechin-3ꞌ-O-rhamnopyranoside.

Figure 2. Effect of compound 1 on N. nigricollis PLA₂ enzyme activity.

Snakebite envenomation is a major public health problem especially in the rural areas of the savannah region of West Africa notably Nigeria, Ghana among others; the African cobras (Naja spp) is one of the major venomous snakes frequently causing envenomation in the northern part of Nigeria [7]. Cobra bites is accompanied by sharp pain (which spread within few minutes), blisters, necrosis, tenderness, inflammation, and severe bacterial infection to mention but few [47–48]. Several bioactive compounds have been evaluated for their antisnake venom [16,49] and other effects. However, to the best of our knowledge, there is no report yet on the antivenom, analgesic, anti-inflammatory and antimicrobial properties of (+)-ZCatechin-3ꞌ-O-rhamnopyranoside. In this study, the effect of compound 1 was evaluated on snakebite, pain, inflammation and microbial infection was investigated.

The survival rate of mice injected with the pre-incubated mixture of an equivalent of the LD₅₀ of N. nigricollis venom and the isolated compound 1 was used to deduce the antivenom effect of the plant [30]. The findings indicated that compound 1 gave maximum protection against N. nigricollis venom-induced actions in a dose-dependent manner with the lowest dose having the highest effect; the possible explanation to this phenomena could be due to saturation at the higher doses. Catechin and gallocatechin were reported to have antivenom effect [50].

Phospholipase A₂ enzyme assay conducted using acidimetry [33]; compound 1 was able to inhibit the action of N. nigricollis PLA₂ enzyme activity with 33.7% inhibition which was lower compared to the standard ASV (72.9%).

Studies have shown that PLA₂ inhibitors constitutes structural features similar to those of phenolic compounds with aromatic ring substituted by one or more hydroxyl groups [51]; which include, phenolic hydroxyl group on carbon 5, the pyronic carbonyl, the double bond, and double-bonded oxygen in the oxane ring [52–53]; for selective inhibition of PLA₂-II, hydroxyl groups in positions 3ʹ and 4ʹ are required (these features are important for the overall capacity of a compound to inhibit PLA₂ activity) [54–55]. Lower effect of compound 1 might be due to the absence of the pyronic carbonyl, the double bond, and double-bonded oxygen in the oxane ring of most flavonoids; however, the sugar moiety might contribute to the observed effect of compound 1 [16].

Formalin acts via two mechanisms in which the first phase involves neurogenic pain that is initiated when harmful stimuli agitate nociceptors while the second phase involves anti-inflammatory reactions [56]. Studies have shown that drugs that inhibit both phases possessed central acting effect and peripheral acting drugs only inhibit the second phase. The ability of the compound to diminish the nociceptive response induced by formalin in both phases suggests that compound 1 may possess central acting analgesic effect with shorter onset and duration of action [56–58].

Formalin is a pro-anti-inflammatory agent that acts by inhibiting acute inflammatory mediators [59]. Formalin-induced inflammation is believed to be biphasic, the early phase (1–2 h) is mediated by histamine, serotonin and increased synthesis of prostaglandins while the late phase involves the release of prostaglandins and it is mediated by bradykinin, leukotrienes and related substances [61]. The ability of compound 1 to significantly (p < 0.05) decreased inflammation induced by formalin suggests that the mechanism of action is related to the inhibition of histamine, serotonin, and related substances as well as the inhibition of prostaglandins synthesis [61].

Based on the postulation that mean zone of inhibition diameter >18 mm [62], compound 1 can be said to possess a good broad spectrum of activity. Also, Tang et al. [63–64] reported that compounds with MIC values less than 100 µg/mL are regarded as very good antimicrobial agents. Catechins and sugars have been reported to have good in vitro antibacterial activity [65–68]. Mori et al. [69] suggested that the ring B of the flavonoids may intercalate or form hydrogen bond with the stacking of nucleic acid bases and further lead to inhibition of DNA and RNA synthesis in bacteria while most antifungal agents act by interfering with the production of fungal protein, DNA replication and via different cellular metabolisms [70], hence the compound might act via the same mechanism. S. aureus, E. coli, and Enterococcus spp were the most common pathogens identified in wound infections associated with snakebites [48]; compound 1 has demonstrated good activity against the pathogens.

In conclusion, the results of this study indicated that catechin-3-O-rhamnopyranoside isolated from the ethylacetate soluble fraction of the stem bark of N. macrophylla has demonstrated significant antivenom, analgesic, anti-inflammatory, and antimicrobial activities, thus providing a new theory in the search for better therapeutic agents in the treatment of snakebite, pain, inflammation and microbial infections. To the best of our knowledge, this is the first detailed biological activity of this compound. Thus, further detailed phytochemical studies is still ongoing in our laboratory with a view to isolate more bioactive compound(s) from the plant.

Acknowledgments

We would like to acknowledge the efforts of Mr. Dilip Jagjivan, School of Chemistry and Physics, University of Kwa-Zulu Natal-Durban, South Africa for running the 1D & 2D-NMR spectroscopy; Dr Mika’ila of the Institute of Leather Research, Samaru, Zaria, for rendering assistance and bench space to conduct the antimicrobial studies and Pharmacist Aminu A. Biambo for running the statistical analysis.

Disclosure statement

No potential conflict of interest was reported by the authors.