UHPLC-QTOF-MS/MS metabolites profiling and antioxidant/antidiabetic attributes of Cuscuta reflexa grown on Casearia tomentosa: exploring phytochemicals role via molecular docking

Figures & data

Table 1. Extract yield, TFC and TPC in different extracts from C. reflexa.

Extraction solvents	Extract yield (%)	TPC (mg GAE/g DE)	TFC (mg RE/g DE)
Pure water	16.02 ± 0.09^E	108.46 ± 2.92^F	79.31 ± 1.28^E
20% ethanol	16.98 ± 0.11^E	114.99 ± 1.31^E	81.81 ± 1.45^DE
40% ethanol	18.31 ± 0.20^D	148.16 ± 2.35^D	85.16 ± 0.42^CD
60% ethanol	20.52 ± 0.44^B	165.25 ± 1.24^B	91.06 ± 1.40^B
80% ethanol	21.89 ± 0.18^A	186.19 ± 2.15^A	106.50 ± 1.68^A
100% ethanol	19.25 ± 0.31^C	158.58 ± 2.09^C	87.12 ± 1.16^C

Alphabets ^(A–F) indicate significant difference (p < 0.05) of mean among solvents used for extraction.

Table 2. DPPH and TAP results for different extracts from C. reflexa extracts.

Extracts	DPPH radical scavenging IC50 (μg/mL)	TAP activity (mg AAE/g DE)
Pure water	113.43 ± 3.35^A	156.51 ± 0.75^E
20% ethanolic	102.26 ± 3.58^B	168.06 ± 1.95^D
40% ethanolic	90.35 ± 2.37^C	182.41 ± 2.43^C
60% ethanolic	79.24 ± 1.38^D	199.56 ± 2.18^B
80% ethanolic	60.64 ± 1.74^E	235.96 ± 1.33^A
100% Ethanolic	85.58 ± 1.10^CD	178.85 ± 1.53^C
BHA*	37.33 ± 1.20^F	NA

Data were articulated as mean ± standard deviations. All measurements were carried out in triplicate. Values marked by different letters as superscripts ^(A-F) indicate statistically significant difference of mean among different samples with p < 0.05.

* = Positive control.

NA = Not applicable.

Table 3. The α-glucosidase and α-amylase inhibition activities for different extracts from C. reflexa.

Solvent compositions	Anti-α-amylase activity (IC50 µg/mL)	Anti-α-glucosidase activity (IC50 µg/mL)
100% water	97.11 ± 0.33^A	87.65 ± 1.67^A
20% ethanol	91.77 ± 1.53^B	82.06 ± 1.53^B
40% ethanol	85.35 ± 0.75^C	75.14 ± 1.12^C
60% ethanol	81.07 ± 1.71^D	64.69 ± 1.06^D
80% ethanol	71.84 ± 1.06^E	57.25 ± 1.40^E
100% ethanol	86.46 ± 0.98^C	76.93 ± 1.0^C
Acarbose*	38.95 ± 0.13^F	34.52 ± 0.26^F

Different capital letters ^(A–F) as superscript in each column describe significance difference among different samples at p < 0.05.

* = Positive control.

Figure 1. UHPLC-QTOF-MS/MS chromatogram of 80% HE C. reflexa extract.

Figure 2. Structures and MS/MS spectra of compounds (1–13) identified in 80% C. reflexa HE extract.

Table 4. Phytoconstituents identified in 80% C. reflexa HE extract by UHPLC-QTOF-MS/MS.

No.	Identification	Rt (min)	[M-H]^−(m/z)	Formula	Mass	Other fragment ions (m/z)
1	(2S)-3-(β-D-galactopyranosyloxy)-2-(hexanoyloxy)propyl nonanoate	11.3	491	C24H44O10	492	391, 329, 311, 229, 211, 171
2	4-O-β-D-glucosyl-4-coumaric acid	7.7	325	C15H18O8	326	265, 235, 205, 163, 145, 119
3	7-(α-D-glucopyranosyloxy)-2,3,4,5,6-pentahydroxyheptanoic acid	1.3	387	C13H24O13	388	341, 323, 221, 179, 161, 113
4	13S-hydroxyoctadecadienoic acid	15.9	295	C18H32O3	296	277, 195, 171, 113, 59
5	Astragaloside derivative	19.1	653	C40H46O8	654	447, 285, 78
6	Caffeic acid 3-glucoside	6.1	341	C15H18O9	342	281, 221, 179, 161, 135
7	Caffeic acid derivative	1.3	387	C13H24O13	388	341, 323, 233, 179, 161,131
8	Citric acid	1.9	191	C6H8O7	192	173,155, 131, 129. 127, 111
9	Salicylic acid	9.8	137	C7H6O3	138	93, 75, 65
10	Isorhamnetin-3-O-glucoside	9.5	477	C22H22O12	478	357, 315, 271, 243, 151
11	Oleanolic acid	18.3	455	C30H48O3	456	383, 326
12	Pinellic acid	12.1	329	C18H34O5	330	229, 211, 183, 171
13	Quinic acid	1.3	191	C7H12O6	192	173, 127, 111, 93, 85

Figure 3. Proposed fragmentation pattern of compounds identified from C. reflexa 80% HE extract.

Figure 4. (a). Ribbon diagram for α-glucosidase (homology modeled). The 3 AARs shown as red sphere (Asp349, Glu276 and Asp214) are catalytic triad residues. The possible active sites are indicated in the form of yellow spheres (b) A ribbon diagram indicating eminent residue in stick form (c) 3-D docking pose of standard drug (acarbose) into the active sites of enzyme (d) A ribbon diagram of superposed compounds of current study into the binding sites.

Figure 5. (a–d). The 3-D interaction plot of identified phytochemicals (1–4) into the active sites of α-glucosidase, respectively. Green dotted lines showed hydrogen bonds and purple dotted lines presented π–π stacking interactions. Distances (in Å) of hydrogen bonds are written in blue. While, for π–π stacking interactions it is written in green.

Figure 6. (a–d). The 3-D interaction plot of identified phytochemicals (6, 8, 9–10), respectively, into the active sites of α-glucosidase.

Figure 7. (a–c). The 3-D interaction plot of identified phytochemicals (11–13) into the active sites of α-glucosidase, respectively (d) 3-D interaction plot of acarbose.

Table 5. Binding affinity values (kcal/mol) and ligand interaction pattern revealed by identified phytoconstituents and acarbose toward yeast α-glucosidase.

Compounds	Binding affinity	Interacting residues	Figure No.
1	−9.1927	Asp68, Asp214, Glu276, Arg312, Asp349, Arg439	5a
2	−7.6887	Phe157, Asp214, His348, Arg439	5b
3	−6.5040	His239, Glu304, Asp349	5c
4	−7.1164	His239, Glu276, Ala278, Glu304, Arg312, Asp349, Arg439	5d
6	−6.6989	Asp68, Arg212, Glu276, Asp349, Arg439	6a
8	−5.2940	Arg312, Lys155, Phe157	6b
9	−4.2390	Glu304, Arg312	6c
10	−7.6978	Asp68, Tyr71, Glu276, Arg312, Asp408	6d
11	−5.6445	Arg312, Asp408	7a
12	−7.7870	Asp214, His279, Glu304, Arg312, Arg439	7b
13	−5.6048	Asp68, Asp214, Glu276, His348, Arg439	7c
Acarbose	−9.2756	Asp214, His279, Glu304, Pro309, Arg312, Asp349, Arg439	7d

Figure 8. (a-k). The 3-D interaction plot for identified phytochemicals (1–4, 6, 8–13), respectively. (l) The 3-D interaction plot of acarbose into active sites of PPA.

Table 6. Binding affinity values (kcal/mol) and ligand interaction patterns computed for possible isolated phytoconstituents and acarbose toward yeast α-amylase.

Compounds	Binding affinity	Interacting residues	Figure No.
1	−7.9445	His201, Asp300	8a
2	−6.5686	Arg195, Asp197, Glu233	8b
3	−6.8663	Asp197, Ile235, His299	8c
4	−6.4928	Asp197, Glu233, Ile235, His299	8d
6	−6.2226	Asp197, Glu233	8e
8	−5.1087	Asp197, His299	8f
9	−4.1284	Glu233	8g
10	−6.9494	Trp59, Asp197, Asp300	8h
11	−6.1213	Glu233	8i
12	−6.9533	His201, Glu233, Glu240	8j
13	−5.0957	Asp197, Glu233, His299	8k
Acarbose	−9.5683	Trp59, Gln63, Arg195, Asp197, Lys200, His201, Glu233, Glu240, Asp300, Gly306.	8l

Figure 9. The S₀ charge-density distribution of FMOs in identified bioactives (contour value = 0.035).

Table 7. The computed ground state HOMO energies (E_HOMO, E_HOMO-1), LUMO energies (E_LUMO, E_LUMO+1), HOMO-LUMO-E_gaps and IP (-E_HOMO) in eV for identified bioactives.

Compounds	EHOMO-1	EHOMO	ELUMO	ELUMO+1	Egaps	IP
1	−7.60	−7.42	−0.49	−0.34	6.93	7.42
2	−7.54	−6.50	−2.14	−0.87	4.36	6.50
3	−7.06	−6.65	−1.66	−0.96	4.99	6.65
4	−7.87	−7.47	−0.18	−0.06	7.29	7.47
6	−6.59	−5.40	−2.31	−1.07	3.09	5.40
8	−8.28	−8.12	−0.79	−0.67	7.33	8.12
9	−7.65	−6.68	−1.94	−0.48	4.74	6.68
10	−6.77	−5.98	−1.97	−1.06	4.01	5.98
11	−7.28	−6.34	−0.28	−0.11	6.06	6.34
12	−7.50	−6.80	−0.22	−0.17	6.58	6.80
13	−7.68	−7.46	−0.81	−0.41	6.65	7.46

Figure 10. MEP surface views of studied compounds.