Naglaa M. Ammar*a,
Heba A. Hassana,
Mona A. Mohammedb,
Ahmed Serag*c,
Sameh Hosam Abd El-Alimd,
Heba Elmotasemd,
Mohamed El Raeye,
Abdel Nasser El Gendyb,
Mansour Sobehf and
Abdel-Hamid Z. Abdel-Hamida
aTherapeutic Chemistry Department, National Research Centre, Dokki, Cairo, 12622, Egypt. E-mail: naglaaammar@yahoo.com
bDepartment of Medicinal and Aromatic Plants Research, National Research Centre, Cairo, Egypt
cPharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 11751 Nasr City, Cairo, Egypt. E-mail: ahmedserag777@azhar.edu.eg; ahmedserag777@hotmail.com
dPharmaceutical Technology Department, National Research Centre, El-Buhouth St., Dokki, Cairo, 12622, Egypt
eDepartment of Phytochemistry and Plant Systematics, National Research Center, Dokki, Cairo 12622, Egypt
fAgroBioSciences, Mohammed VI Polytechnic University, Lot 660, Hay MoulayRachid, Ben-Guerir 43150, Morocco
First published on 23rd February 2021
Posidonia oceanica is a sea grass belonging to the family Posidoniaceae, which stands out as a substantial reservoir of bioactive compounds. In this study, the secondary metabolites of the P. oceanica rhizome were annotated using UPLC-HRESI-MS/MS, revealing 86 compounds including simple phenolic acids, flavonoids, and their sulphated conjugates. Moreover, the P. oceanica butanol extract exhibited substantial antioxidant and antidiabetic effects in vitro. Thus, a reliable, robust drug delivery system was developed through the encapsulation of P. oceanica extract in gelatin nanoparticles to protect active constituents, control their release and enhance their therapeutic activity. To confirm these achievements, untargeted GC-MS metabolomics analysis together with biochemical evaluation was employed to investigate the in vivo anti-diabetic potential of the P. oceanica nano-extract. The results of this study demonstrated that the P. oceanica gelatin nanoparticle formulation reduced the serum fasting blood glucose level significantly (p < 0.05) in addition to improving the insulin level, together with the elevation of glucose transporter 4 levels. Besides, multivariate/univariate analyses of the GC-MS metabolomic dataset revealed several dysregulated metabolites in diabetic rats, which were restored to normalized levels after treatment with the P. oceanica gelatin nanoparticle formulation. These metabolites mainly originate from the metabolism of amino acids, fatty acids and carbohydrates, indicating that this type of delivery was more effective than the plain extract in regulating these altered metabolic processes. Overall, this study provides novel insight for the potential of P. oceanica butanol extract encapsulated in gelatin nanoparticles as a promising and effective antidiabetic therapy.
Posidonia oceanica (L.) Delile, from the family Posidoniaceae, is a long-living, slow-growing, endemic Mediterranean seagrass.5 P. oceanica has been called “the lungs of the Mediterranean” as it is one of the most important sources of oxygen in coastal waters. Interestingly, P. oceanica is also characterized by its vast content of phenolic compounds.6 These include the phenols found in P. oceanica leaves such as phenolic acids (4-hydroxybenzoic acid, gentisic acid, chicoric acid, cinnamic acid, ferulic acid, p-coumaric acid, and caffeic acid), flavonoids (kaempferol, quercetin, isorhamnetin and myricetin) and vanillin.7 The medicinal uses of P. oceanica date back to ancient Egypt, where it was used to treat skin diseases, while it has been used recently as a remedy for hypertension, respiratory infections, diabetes, colitis, and acne in the southwestern Mediterranean region.8
Metabolomics, as the metabolic complement of functional genomics, is a promising approach to explore homeostasis. The main goal of metabolomics in diabetes and other diseases is to discover metabolic biomarkers and perturbed pathways, which can be used as tools for medical practice such as the diagnosis and prognosis of therapeutic response including herbal medication.9 Recently, with the development of metabolomic technology, more reliable control of diabetes mellitus is expected to be achieved based on a patient's metabolomic profile.
The aim of the present study was to develop a metabolomic-based approach for characterizing the secondary bioactive polyphenolic metabolic pool of P. oceanica using UPLC-HRESI-MS/MS. Moreover, a reliable, robust drug delivery system was developed by encapsulating P. oceanica extract in gelatin nanoparticles to increase its bioactivity and therapeutic potency. The in vitro characterization of this novel nano-extract was performed. In addition, evaluation of its in vivo antidiabetic activity compared to the plain extract was conducted in a comparative way via GC-MS-based metabolomics together with different biochemical tests.
For absolute quantification of some secondary metabolites according to the availability of their analytical standards, an HPLC system (HP 1100 chromatograph, Agilent Technologies, Palo Alto, CA, USA) equipped with an auto-sampler (G1329B), quaternary pump and a diode array detector was employed. Separation was achieved using a ZORBAX Eclipse XDB C18 column (15 cm × 4.6 mm I.D., 5 μm, USA) under the following conditions: mobile phase A, 2% acetic acid; mobile phase B, acetonitrile; flow rate, 0.8 mL min−1; fixed wavelength, 280 and 360 nm; injected quantity, 10 μL; elution program, A (%)/B (%): 0 min 90/10; 15 min 50/50; 17 min 20/80; 19 min 90/10; and 20 min 90/10. Identification of phenolic compounds was performed by comparison with the retention times of standard substances.
No. | RT [min] | Metabolite identification | Chemical formula | [M − H]− | Relative abundance (%) | |||
---|---|---|---|---|---|---|---|---|
Measured, calculated | Fragmentation | MeOH | BuOH | EtOAc | ||||
a Compounds isolated before from the plant.b Quantitative estimation of some identified compounds (μg g−1) using/HPLC-DAD.c Data was collected in the positive-ion mode. * indicate the presence of a metabolite in the selected fraction. | ||||||||
1 | 1.55 | Citric acid | C6H8O7 | 190.9278, 191.0270 | 162.89240, 146.93741, 111.00736 | * | * | |
2 | 2.00 | 4-Hydroxybenzoylcholinea | C12H17O3N | 224.1281c, 224.1281 | 165.049, 155.9746, 125.2647 | * | ||
3 | 2.22 | Monogalloyl glucose | C13H16O10 | 331.0671, 331.0744 | 169.0132 | * | * | |
4 | 2.2 | Succinic acid | C4H6O4 | 177.0179, 117.0182 | 199.0072, 86.1084, 73.0279 | * | * | * |
5 | 2.28 | Malic acid | C4H6O5 | 133.0129, 133.0215 | 115.0022, 89.0229 | * | ||
6 | 2.29 | Quinic acid | C7H12O6 | 191.0553, 191.0634 | 133.0129, 111.0073 | * | * | |
7 | 2.36 | 5-Methoxypsoralen | C12 H8 O4 | 215.0230, 215.0339 | 113.0224, 89.0228, 71.0123 | * | * | |
8 | 2.47 | Phloracetophenone | C8H8O4 | 167.0201, 167.0200 | 124.0138, 118.2005, 96.086 | * | ||
9 | 2.47 | Hordenine | C10H15NO | 166.1226c, 166.226 | 148.0484, 121.0651, 107.0495 | * | ||
10 | 2.94 | Danshensu | C9H10O5 | 197.8073, 197.0449 | 162.8379, 103.9188, 122.8925 | * | * | |
11 | 3.35 | Benzoylcholine | C12H17NO2 | 208.1333c, 208.1305 | 149.0596, 131.0497, 105.0336 | * | ||
12 | 3.86 | Phloroglucinol | C6H6O3 | 125.0231, 125.0233 | 97.0280, 81.0331, 69.0330 | * | ||
13 | 3.93 | P-Hydroxyl benzoic acid | C13H16O8 | 299.0775 | 137.0231 | * | * | |
14 | 3.98 | Protocatechuic acida,b | C7H6O4 | 153.0182, 153.0266 | 109.0281 | *11.02 | *12.01 | *8.51 |
15 | 3.99 | Catechola | C6H6O2 | 109.0281, 109.0284 | 92.8299, 81.0330, 65.0380 | * | * | * |
16 | 4.00 | Protocatechuic acid hexoside | C13H16O9 | 315.0724, 315.0711 | 279.8815, 153.0183, 123.0437 | * | ||
17 | 4.04 | Methyl salicylatea | C8H8O3 | 151.0023, 151.0026 | 136.0155, 123.0074, 107.0124 | * | ||
18 | 4.48 | Vanillina | C8H8O3 | 151.0391, 151.0473 | 123.0074, 107.0123 | * | * | * |
19 | 4.06 | Gallic acida,b | C7H6O5 | 169.0133, 170.0215 | 125.0231 | *3.77 | *3.70 | |
20 | 4.41 | Vanillic acid hexoside | C14H18O9 | 329.0883, 329.0951 | 167.0341, 119.0338 | * | * | |
21 | 4.45 | Homovanillic acid hexoside | C15H20O9 | 343.1039, 343.1107 | 181.0495 | |||
22 | 4.46 | Galloyl shikimic acid | C14H14O9 | 325.0569 | 169.0134, 125.0230 | * | * | |
23 | 5.03 | Hydroxybenzoic acid pentoside | C12H14O7 | 269.0671, 269.0656 | 137.0232 | * | ||
24 | 5.05 | Hydroxyferulic acid | C10H10O5 | 209.0450, 209.0528 | * | |||
25 | 5.06 | P-Hydroxybenzoic acidb | C7H6O3 | 137.0231, 137.0317 | 93.0331 | *56.52 | *64.04 | *30.26 |
26 | 5.08 | P-Coumaric acidb | C9H8O3 | 162.838 | 119.0486 | *3.27 | *0.42 | *5.71 |
27 | 5.08 | Salicylic acida | C7H6O3 | 137.0231, 137.0233 | 107.0029, 93.0331, 65.0381 | * | ||
28 | 5.09 | 4,6-Dihydroxy-3-(1-hydroxyethyl)-5-methoxy-2-benzofuran-1(3H)-one | C11H12O6 | 239.0546, 239.0546 | 221.0453, 192.9584, 165.0183 | * | ||
29 | 5.12 | Protocatechuic acid sulphate | C7H6O7S | 232.976, 232.9756 | 153.0183 | * | * | |
30 | 5.14 | Caffeic acid hexoside | C15H18O9 | 341.1107, 341.1957 | 179.0551, 161.0442, 119.0335, 89.02283 | * | ||
31 | 5.26 | Benzoic acida | C7H6O2 | 121.0281, 121.0284 | 108.0204, 93.0331, 75.7648 | * | * | |
32 | 5.56 | 4-Deoxyphloridzin | C21H24O9 | 419.1400, 419.1395 | 257.0854, 271.1304, 196.0937 | * | ||
33 | 5.35 | Catechinb | C15H14O6 | 289.0716, 289.0707 | 245.0130, 179.0342, 125.0229 | *0.01 | *8.20 | *0.1 |
34 | 5.2 | Daphnetin | C9H6O4 | 177.0184, 177.0182 | 162.0320, 133.0281, 105.0330 | * | ||
35 | 5.56 | Syringaldehydea | C9H10O4 | 181.0134, 181.0131 | 166.0264, 137.0232, 125.0178, 98.0331 | * | * | |
36 | 5.63 | 3-Methylcatechol | C7H8O2 | 123.0436, 123.0441 | 108.0202, 95.0125, 72.9686, 68.3135 | * | ||
37 | 5.64 | Procyanidin B2a | C30H26O12 | 577.1420, 577.1420 | 557.2190, 407.0811, 289.0718 | * | ||
38 | 5.7 | Ethyl cinnamate | C11H12O2 | 174.9552, | 160.9757, 146.9598, 130.9424, 118.946, | * | ||
39 | 5.67 | Caffeic acid dimethyl ester | C11 H12O4 | 207.0656, 207.0652 | 179.9351, 159.858, 127.868, 103.918, 87.9237 | * | ||
40 | 5.79 | Gentisic acidb | C7H7O4 | 154.0248, 154.0261 | 122.8927, 109.0280, 110.031396.9586, 59.5949 | * | ||
41 | 5.85 | Kynurenic acid | C10H8NO3 | 188.0343, 188.0342 | 144.0443, 109.0278 | * | ||
42 | 5.89 | Homovanillic acida | C9H10 O4 | 181.0493, 181.0495 | 153.0181, 137.0231, 123.0437, 109.0280 | * | ||
43 | 6.16 | Homo gentisic acida | C8H8O4 | 167.0339, 167.0339 | 123.0072, 108.0199, 95.0124 | * | ||
44 | 6.19 | Epi-catechina | C15H14O6 | 289.0392 | 245.082 | *0.54 | *10.06 | |
45 | 6.25 | Vanillic acid 4-sulfate | C8H8O7S | 246.9914, 246.9262 | 167.0340, 159.0441 | * | ||
46 | 6.30 | Dihydroluteolin-O-hexoside | C21H22O11 | 449.1089, 449.1162 | 287.0563, 269.0458, 178.9978 | * | * | * |
47 | 6.38 | Ferulic acid hexoside | C16H20O9 | 355.1040, 355.1107 | 193.0496 | * | ||
48 | 6.39 | Protocatechuic aldehyde | C7H6O6S | 216.9805, 216.9801 | 199.8505, 159.8586, 172.99701, 137.02315, 119.9765 | * | ||
49 | 6.58 | Catechin gallate | C22H18O10 | 441.1247, 441.1239 | 169.6788, 171.9455, 160.8407, 123.2177 | * | * | |
50 | 6.66 | Ferulic acidb | C10 H10O4 | 193.0497, 193.0495 | 177.0545, 149.5339, 90.9320 | *0.47 | *4.42 | *0.38 |
51 | 6.7 | Coumaroyl quinic acid | C16 H18O8 | 337.0918, 337.0923 | 322.0846, 191.0705, 147.0801, 119.6231 | * | ||
52 | 6.76 | Coniferyl aldehydea | C10H10O3 | 177.1633, 177.1638 | 167.0339, 152.0103, 124.0152, 111.0074 | * | ||
53 | 6.69 | Sinapic acida,b | C11H12O5 | 223.0610, 223.0685 | 179.0706, 123.438 | *0.11 | *2.76 | |
54 | 6.92 | Vanillic acidb | C8 H8O4 | 167.0339, 167.0339 | 152.0104, 123.0074, 111.0073, 66.463 | *6.43 | *1.38 | *12.51 |
55 | 6.96 | Salvaianolic acid G | C18H12O7 | 399.2004 | 321.15760, 66.8679 | * | ||
56 | 7.06 | Quercetin-O-pentoside-O-rhamnoside | 597.2015 | 417.1549, 327.0145, 213.4272, 181.0498 | * | |||
57 | 7.34 | Cinnamic acida,b | C9H8O2 | 147.0440, 147.0441 | 123.9449, 102.9473, 87.9238, 61.9867 | *0.50 | *2.98 | *0.68 |
58 | 7.35 | Syringic acida,b | C9H10O5 | 197.8072, 197.8073 | 162.8380, 123.9005, 103.9186 | *1.49 | *9.83 | *15.01 |
59 | 7.37 | Gallocatechin | C15H14O7 | 305.0699, 305.0715 | 225.1128, 169.7713, 138.7856 | * | ||
60 | 7.52 | p-Coumaraldehydea | C9H8O2 | 147.0439, 147.0441 | 129.0334, 119.0490, 102.9473 | * | ||
61 | 8.09 | Gallocatechin gallate | C22H18O11 | 457.0765, 457.0771 | 137.0958, 123.0438, 114.9323 | * | * | |
62 | 8.19 | Scopoletinb | C10H8O4 | 191.0338, 191.0339 | 162.8923, 146.9375, 111.0073 | *0.52 | *2.61 | |
63 | 8.27 | Procyanidin A2a | C30H24O12 | 575.1262, 575.1300 | 407.0793, 161.8318, 125.0228 | * | ||
64 | 8.61 | Caffeic acida,b | C9H8O4 | 179.0343, 179.0339 | 150.9530, 134.9868, 90.9966 | *1.34 | *1.01 | *0.56 |
65 | 8.64 | Ellagic acida | C14H6O8 | 300.9992, 300.9979 | 257.0097, 185.0247 | * | ||
66 | 8.68 | 2′-Hydroxygenistein-7-O-glucoside | C21 H19O11 | 447.0944 | 317.1527, 285.0407, 134.4610, 103.1963 | * | ||
67 | 8.85 | Rutinb | C27H30O16 | 609.1588, 609.1589 | 301.0715, 173.603, 75.0797 | *9.33 | *0.68 | *1.39 |
68 | 8.99 | Kaempferol-3-glucuronide | C21 H18 O12 | 461.0737, 461.0715 | 403.9498, 285.0404, 148.9581 | * | ||
69 | 9.09 | Secoisolariciresinol | C20H27O6 | 361.1659, 361.1646 | 346.1428, 331.0833, 179.0741, 137.0230 | * | ||
70 | 9.39 | Rosmarinic acidb | C18H16O8 | 359.1136, 359.1125 | 257.0820, 197.0451, 179.036, 161.0233 | *0.08 | *0.02 | *1.14 |
71 | 9.46 | Pyridinesulfonamides | C16H20N4O3S | 347.1172, 347.1175 | 274.4805, 137.0233, 195.0652, 162.0544 | * | ||
72 | 9.51 | Apigenin-7-O-glucosideb | C21H20O10 | 431.0973, 431.0975 | 321.6589, 268.038, 79.8678 | *0.55 | *2.30 | *0.01 |
73 | 9.72 | Luteolin-5-glucoside | C21H20O11 | 447.1045, 447.1028 | 314.0438, 271.0258, 151.0024, 89.0907 | * | ||
74 | 9.86 | Baicalein-7-O-glucuronide | C21H18O11 | 445.0786, 445.0765 | 269.0458, 151.0388, 113.0232 | * | ||
75 | 10.54 | P-Anisic acida | C8H8O3 | 151.0388, 151.0390, | 136.0153, 123.0074, 107.0488, 93.0331 | |||
76 | 10.95 | Gibberellin A42 | C20H30O6 | 365.1975, 365.1959 | 335.0222, 267.0664, 255.0665, 166.6308 | * | ||
77 | 11.53 | Enterolactone | C18H18O4 | 297.1132, 297.1211 | 253.1231, 189.0550, 107.0488 | |||
78 | 11.71 | Luteolin b | C15H10O6 | 285.0406, 285.0394 | 250.4250, 151.0027, 137.0230, 93.0329 | *3.72 | *0.01 | *0.03 |
79 | 11.85 | Naringeninb | C15H12O5 | 271.0613, 271.0601 | 253.1438, 151.0023, 119.0487 | *0.23 | *3.13 | *2.49 |
80 | 12.95 | Gibberellic acid A44 | C20H26O5 | 345.1709, 345.1780 | 301.1812, 283.1712 | * | ||
81 | 13.15 | Diosmetin | C16H12O6 | 299.0565, 299.0550 | 284.0327, 237.1862, 125.0957 | * | * | |
82 | 13.36 | Kaempferolb | C15H10O6 | 285.0406, 285.0394 | 254.9923, 183.9123, 137.0231 | |||
83 | 14.13 | Butylparaben | C11H14O3 | 193.0862, 193.0859 | 178.0257, 137.0231, 106.0755 | * | ||
84 | 17.51 | Apigeninb | C15H10O5 | 269.0457, 269.0444 | 251.2011, 189.0912, 155.1069 | *0.67 | *0.01 | *0.91 |
85 | 17.62 | Methylated (−)-gallo catechin gallate | 471.0768 | 431.8876, 385.5272, 341.1092, 325.1846, 245.9779, 169.9367, | * | |||
86 | 18.92 | Bilobalide | 325.1844 | 250.1201, 183.0110, 108.1780 | * |
Fig. 1 UHPLC-MS traces of P. oceanica extracts showing different qualitative differences according to their metabolic profiles. |
Interestingly, the highest concentrations of total phenols and flavonoids were also found in the butanol fraction, which is consistent with the HPLC-DAD quantification results. Also, the in vitro antidiabetic activity agreed with the total phenol and flavonoid content and the antioxidant activities using the DPPH assay, as shown in Table 2.
Extract | Phenolic content (mg gallic acid/g extract) | Flavonoid content (mg rutin/g extract) | DPPH (IC50, μg mL−1) | α-Glucosidase (IC50, μg mL−1) |
---|---|---|---|---|
BuOH | 200.20 ± 1.09 | 40.17 ± 0.11 | 10.5 | 4.8 ± 0.3 |
EtOAc | 140.25 ± 1.01 | 20.34 ± 0.27 | 30.4 | 8.9 ± 0.4 |
Total alcohol | 120.37 ± 1.17 | 20.35 ± 0.03 | 76.3 | 24.8 ± 2 |
Acarbose (SD) | — | — | — | 4.5 ± 0.27 |
Code | Gelatin (g) | Stabilizer | Gelatin: stabilizer ratio (w/w) | Solvent, water (mL) | Non-solvent, ethanol (mL) | NP formed | EE% ± SD | PS (nm) ± SD | ZP (mV) | |
---|---|---|---|---|---|---|---|---|---|---|
Poloxamer 407 (g) | Poloxamer 188 (g) | |||||||||
G1 | 0.2 | 1.6 | 0 | 1:8 | 10 | 80 | + | 51.68 ± 2.34 | 274.7 ± 30.5 | −10.7 |
G2 | 0.2 | 3.2 | 0 | 1:16 | 10 | 80 | + | 63.76 ± 2.38 | 290.0 ± 13.66 | −12.3 |
G3 | 0.2 | 6.4 | 0 | 1:32 | 10 | 80 | + | 51.68 ± 2.34 | 274.7 ± 30.5 | −10.7 |
G4 | 0.2 | 1.6 | 0 | 1:8 | 10 | 40 | − | — | — | — |
G5 | 0.2 | 3.2 | 0 | 1:16 | 10 | 40 | + | — | — | — |
G6 | 0.2 | 6.4 | 0 | 1:32 | 10 | 40 | + | — | — | — |
G7 | 0.2 | 0 | 1.6 | 1:8 | 10 | 80 | + | 32.65 ± 5.39 | 591.1 ± 37.97 | −10.0 |
G8 | 0.2 | 0 | 3.2 | 1:16 | 10 | 80 | + | 41.80 ± 2.12 | 461.1 ± 47.83 | −10.5 |
G9 | 0.2 | 0 | 6.4 | 1:32 | 10 | 80 | + | 49.48 ± 3.59 | 458.7 ± 53.95 | −14.8 |
G10 | 0.2 | 0 | 1.6 | 1:8 | 10 | 40 | − | — | — | — |
G11 | 0.2 | 0 | 3.2 | 1:16 | 10 | 40 | − | — | — | — |
G12 | 0.2 | 0 | 6.4 | 1:32 | 10 | 40 | − | — | — | — |
The EE% of the prepared gelatin nanoparticle formulations is reported in Table 3. It was obvious that higher EE% values were observed when Poloxamer 407 was used as a stabilizer in comparison to Poloxamer 188. These results also revealed that the increment in EE% values was directly proportional to the increase in stabilizer ratio in all the investigated formulations. This finding was more pronounced when employing high stabilizer to gelatin ratios (G2 and G3 in comparison to G8 and G9). Moreover, all the investigated formulations showed particle size values in the nanosize range (242–591 nm) (Table 3). The zeta potential of the gelatin nanoparticles exhibited negative values in the range of −10.0 to −14.8 mV, reflecting moderate physical stability for the nanoparticulate formulations. These results are in accordance with previous reports on gelatin nanoparticles.28,29 Based on the results of EE%, PS and ZP, formulation G3 was found to exhibit the highest EE% and suitable PS and ZP values, and thus was selected for further investigations. Besides, TEM was employed to elucidate the shape and morphology of the selected nanoparticle formulation, G3. Fig. 2 shows that the nanoparticles are homogeneous and appear as dark stained spheroid shapes with no signs of aggregation.
Fig. 3 illustrates the release profile of the polyphenols from the selected gelatin nanoparticle formulation, G3. The depicted release profile was gradual, which extended over 24 h, indicating that encapsulating the extract within the gelatin nanoparticle formula was successful in affording a controlled release pattern in the GIT pH mimetic condition. There was no detectable abrupt burst release for the polyphenols. The cumulative percentage release amount was satisfactory, reaching about 20% within 2 h at pH 1.2. This was followed by a slower gradual rate of release at the intestinal pH of pH 6.8, attaining about 36% after 6 h. Subsequently, further amounts of the encapsulated polyphenols were released within the subsequent 18 h at pH 7.4, where the total cumulative amount of released phenolics reached nearly 70% within 24 h.
The kinetic study of the release data disclosed that both the diffusion (Higuchi) model and first-order model depicted high regression coefficient values (0.98 and 0.97, respectively). Further, fitting 60% of the release data to the Peppas model revealed that the “n” value was 0.46, thus lying in the range between 0.43 and 0.85, indicating an anomalous non-Fickian release pattern.20 This confirmed that different contributing factors dominated the release of the polyphenols from the gelatin nanoparticles.30
Groups | FBG (mmol L−1) | INS (μIU mL−1) | HOMA-IR | GLUT 4 (ng mL−1) |
---|---|---|---|---|
a P < 0.05 versus diabetic rats.b P < 0.05 versus normal control. | ||||
Normal control | 4.79 ± 0.12a | 11.23 ± 0.58a | 2.45 ± 0.14a | 11.66 ± 0.12a |
Diabetic rats | 21.41 ± 0.45b | 43.13 ± 4.67b | 41.38 ± 3.67b | 8.61 ± 0.44b |
Plain extract treated group | 12.10 ± 1.25a,b | 35.03 ± 4.48b | 20.38 ± 3.78a,b | 8.42 ± 0.38b |
Standard drug treated group | 11.86 ± 0.92a,b | 26.30 ± 4.05a | 13.10 ± 2.07a | 9.17 ± 0.66b |
Nano-extract treated group | 7.88 ± 0.44a | 13.50 ± 2.14a | 4.82 ± 0.96a | 11.27 ± 0.42a |
A metabolomics approach based on GC-MS was also developed to investigate the antidiabetic activity of P. oceanica in rats. A total of 207 characteristic m/z features were detected in this study, among which 72 annotated metabolites survived the QC-based filtering procedures. Data normalization by sum of total intensities for each sample was carried out to make the metabolite intensity more comparable. Besides, a combination of log transformation and Pareto scaling yielded a Gaussian distribution for the data. This normality of the data enabled the application of different multivariate and univariate tests to examine the trends among the studied groups to reveal the significant metabolites associated with the antidiabetic activity of P. oceanica and its nano-formulation. Twenty-five differential metabolites were revealed from the statistical analysis of the GC-MS data, which belonged to different classes, including organic acids, amino acids, sugars and fatty acids. The identities, chromatographic and mass spectrometry data of these metabolites are listed in Table 1S.†
PCA was carried out to visualize the patterns among the different studied groups in an unsupervised manner. The scores of the control group and groups treated with P. oceanica gelatin nanoparticles and glibenclamide showed a separation trend along the first projection with positive score values from the diabetic and the butanol-treated groups with negative score values, but further differentiation was not obvious (Fig. 4). Hence, PLS-DA as a supervised chemometric approach was employed to derive better sample classification owing to its capability to identify correlation with the phenotypic variable of interest. The PLS-DA score plot showed clearer discrimination among sample groups compared to PCA, with an R2 value (0.66) and Q2 value (0.52) using three latent component models (Fig. 5a). The PLS-DA class inner relationship model of all the groups revealed that the diabetic and P. oceanica butanol-treated group rats were located the furthest from normal rats and other treatment groups (Fig. 5b). Remarkably, the P. oceanica gelatin nanoparticle-treated group was the closest to the control normal rats, suggesting that this line was the most effective treatment approach to restore the metabolite profile in the diabetic rats, mirroring that in the normal rats. The validity of the developed PLS-DA model was confirmed by performing a permutation test (1000 times) with pR2Y and pQ2 (>0.001) for the multi-group comparison. The metabolites contributing this group segregation were revealed by calculating the variable influence of projection (VIP) together with their regression coefficients (Table 2S†). They include beta-hydroxybutyric acid, glucosamine, butane-2,3-diol, glucose, 1,5-anhydro-D-glucitol, aminobutyric acid, myo-inositol and GABA.
OPLS-DA is another supervised approach that is superior to other multivariate methods in terms of discriminating ability and biomarker discovery owing to its capability to remove the non-predictive variations through orthogonal signal correction. However, only pairwise comparison can be implemented using this model, and consequently, different OPLS-DA models were constructed to reveal the pharmacometabolomic effect of P. oceanica and its nano-extract. The R2Y and Q2Y values were used to determine the quality of each OPLS-DA model and evaluate its prediction power. For the control group versus the streptozotocin-induced diabetic group, the model validation parameters were: R2 = 0.958 and Q2 = 0.674 with clear separation along the predictive component, as shown in Fig. 6a. Another separation trend was depicted in Fig. 6b from modelling the P. oceanica butanol-treated group versus the diabetic samples with R2 = 0.952 and Q2 = 0.721. Moreover, a final model was built comparing the P. oceanica (L.) extract encapsulated in gelatin nanoparticle-treatment group versus their diabetic counterparts. The score plot of this model as depicted in Fig. 6c shows a clear separation with R2 = 0.928 and Q2 = 0.774. Additionally, all the OPLS-DA models were strictly validated using permutation tests (1000), where all the Q2 values of the permuted data set show a significant model with pQ2 (0.001 each) for pairwise comparison of the diabetic rats versus the control, P. oceanica butanol- and nano-extract treatment groups. Besides, the S plot loadings, a useful tool generated for each OPLS-DA model by plotting the covariance (p) against correlation p(corr), was implemented to decipher the relevant biomarkers contributing to the detected differences, as shown in Fig. 6d–f. The results revealed some metabolites with higher p(corr) values, accounting for the higher expression in diabetic rats compared to the control and P. oceanica encapsulated in gelatin nanoparticles treatment groups including glucose, alanine, leucine, isoleucine, proline, tyrosine, phenylalanine, oleic acid and linoleic acid.
Fig. 6 GC-MS-based OPLS-DA score plots derived from modelling diabetic rats versus other groups (A–C). The scores of the samples are coded as follows: Gp 1, healthy control group; Gp 2, non-treated diabetic group; Gp 3, diabetic group treated with plain butanol extract of P. oceanica; Gp 4, diabetic group treated with the nano-extract of P. oceanica. Derived S-plots (D–F) showing the covariance p[1] against the correlation p(cor)[1]. Selected variables follow that listed in Table S2† for metabolite identification: S2; butane-2,3-diol, S3; lactic acid, S4; alanine, S11; proline, S13; serine, S17; phenylalanine, S19; glucose, and S22; tyrosine. |
However, for a better selection of the differential metabolites, a combined univariate and multivariate strategy was developed considering the VIP of the OPLS-DA models together with the univariate t-test statistical analysis and metabolite fold change (Table 3S†). The variables that influence the projection (VIP) >1.3 with a q value <0.05 and normalized fold changes >2 or <0.5 were selected. In agreement with the OPLS-DA-derived S plots, the nano P. oceanica treatment group has the highest number of metabolites, among which, their relative concentrations were statistically different from the diabetic group [13 metabolites belonged mainly to amino acids, fatty acids and sugars] followed by the control group [8 metabolites] and butanol P. oceanica treatment group [5 metabolites]. The fold change of the diabetic rats divided by the normal control and nano P. oceanica treatment groups suggested a significant increase in butane-2,3-diol, alanine, leucine, isoleucine, proline, tyrosine, phenylalanine, methionine, 2-hydroxyhexanoic acid, oleic acid and linoleic acid (Table 3S†). However, the butanol P. oceanica treatment group failed to decrease the relative concentration levels of these metabolites.
To provide a gradual controlled release that provides protection of these polyphenols against degradation in the harsh GIT condition especially, the butanol fraction was formulated in a nano form. Gelatin nanoparticulate formulations were prepared employing the nanoprecipitation technique. In this process, nanoparticle formation is based on the interfacial turbulence generated by solvent displacement from the internal phase.36 Successful preparation was observed when a higher solvent/non solvent ratio was used (1:8 v/v). The solvent/non-solvent ratio is an important parameter for preparing stable nanoparticles,37 where higher ratios lead to the production of small stable nanoparticles. It is also worth noting that unlike Poloxamer 188, Poloxamer 407 had the ability to form stable nanoparticles when higher concentrations were employed.
The increase in EE% indicates that the stabilizing effect of poloxamers on the gelatin nanoparticles is concentration dependent.37 Higher EE% values and smaller particle sizes were observed when Poloxamer 407 was used as the stabilizer compared to Poloxamer 188. This observation was more pronounced when higher concentrations of the stabilizer were used and can be attributed to the higher molecular weight of Poloxamer 407 (12000 g mol−1) compared to Poloxamer 188 (8350 g mol−1). It was previously reported that stabilizers having a lower molecular weight failed to produce the required stabilizing effect.37
The in vitro release profile of the selected formulation, G3, showed that the cumulative polyphenol amount released at the intestinal region pH (6.8–7.4) was larger than that released at the acidic stomach pH. This can be attributed to the gelatin type used in the nanoparticle preparation (type B), which will become negatively charged at near neutral pH values, thus causing greater swelling and permitting a larger amount of the encapsulated drug to be released in comparison to acidic pH.38 This gradual controlled release provides protection to the polyphenols against degradation in the harsh environment in the stomach region. Also, it will permit a larger amount of polyphenols to reach their target site considering that it has been reported that the intestinal region is the major site of polyphenol absorption.39,40
This investigated formulation (G3) was further subjected to in vivo evaluation in diabetic rats to estimate the implication of encapsulation in the gelatin nanoparticles on promoting the therapeutic activity of P. oceanica extract. Therefore, induction of diabetes was first performed and assessed by measuring the levels of fasting blood glucose, which showed a significant increase in the STZ hyperglycemic rats due to the destruction of glucose homeostasis.41 The muscle and fat cells are “resistant” to insulin action in type 2 diabetes (T2DM) and compensatory mechanisms are triggered in the β-cells to secrete more insulin.42 Therefore, the HOMA-IR levels in diabetic rats were found to be significantly higher than that in normal control rats. However, these levels decreased significantly after treatment with P. oceanica extract encapsulated in gelatin nanoparticles for 28 days, suggesting that the P. oceanica extract encapsulated in the gelatin nanoparticles is capable of reducing insulin resistance and improving the sensitivity of the body to insulin.
Additionally, there was a significant reduction in the level of Glut 4 in the diabetic rats compared to the normal control, in agreement with Sukanya et al.,43 who reported that the level of Glut 4 was significantly reduced in diabetic rats by mediating glucose uptake and regulating the transport of glucose into muscle cells. Interestingly, the P. oceanica extract encapsulated in gelatin nanoparticles also showed substantial improvement in the level of Glut 4 and glucose homeostasis.
The serum metabolic profiling of the control and T2DM treatment groups was also investigated to explore the potential serum biomarkers and changes in the metabolic pathways involved in the treatment of diabetic rats with the P. oceanica butanol fraction in both its free form and encapsulated in gelatin nanoparticles (Fig. 7). As predicted, the hexose level in our present study was strongly correlated with T2DM, where hexose involves not only glucose, but all six-carbon monosaccharides. Pancreatic beta-cell dysfunction and insulin resistance can be implied by an elevated hexose level. Our result is consistent with the results of other studies,44,45 which confirmed that hexose metabolites are relevant for the assessment of T2DM associations. The present results indicated a significant reduction in the blood glucose level in the diabetic rats pretreated with P. oceanica extract and that encapsulated in the gelatin nanoparticle formula was more effective. This finding suggests that encapsulation in the form of nanoparticles is highly effective in reducing the endogenous glucose production (EGP) metabolite level in diabetic rats. This study also demonstrated that many saccharide species related to glucose metabolism were notably decreased in the group treated with the P. oceanica nanoparticle formulation (G3). The reduced saccharide species in this group implied the improvement of glucose metabolism, leading to depletion in the risk of disease complications.
Furthermore, the diabetic group depicted a significant increase in the concentration of BCAAs (valine, leucine, and isoleucine), proline, methionine, phenylalanine, alanine and tyrosine (Table 3S† and Fig. 7) in comparison to the normal control group. These results are in agreement with that of Zhang et al.,1 who reported that an elevation in the level of branched-chain amino acids actually leads to insulin resistance by declining the activity of AMP-activated protein kinase, which finally leads to T2DM.46 Moreover, increased BCAAs elicit catabolic substances (propionyl CoA and succinyl CoA), resulting in the aggregation of incompletely beta-oxidized fatty acids and glucose, decreased insulin effect, and glucose control disorder.45 It was observed that this increase was significantly reversed by the P. oceanica extract encapsulated in gelatin nanoparticles. The beneficial effects of P. oceanica butanol extract on the enhanced metabolism of amino acids can be clarified by its beneficial impact on circulating insulin concentrations and insulin sensitivity by activating the cAMP/PKA-dependent ERK1/2 signaling pathway.47
It was also found that aromatic amino acids such as phenylalanine and tyrosine were perturbed in the diabetic group, which is consistent with previous metabolomic studies of T2DM.48,49 Asserted pathways that relate this association include inhibiting the transport/phosphorylation of glucose50 and enhancing of insulin resistance by phosphorylation of insulin receptor substrate 1.51 Phenylalanine and tyrosine were reported to be related to insulin resistance, which confirms their roles in the pathogenesis of T2DM.52
The elevation of the sugar alcohol butane-2,3-diol concentrations suggests dysregulation of glucose homeostasis and stimulation of specific pathways such as polyol metabolism (Fig. 7). Indeed, sugar alcohols can also be derived from microbiota and food metabolism in the gut due to impaired intestinal barriers,53 and thus the attenuated level of butane-2,3-diol in the group treated with the P. oceanica extract encapsulated in gelatin nanoparticles suggests the regulation of glucose homeostasis.
Increased oleic and linoleic acids levels were also observed, which displayed lipotoxicity in the rats treated with STZ. This is consistent with the findings of Lu et al.,54 who reported that fatty acids inhibit insulin action via the Randle cycle, intracellular lipid derivative accumulation, inflammation, oxidative stress and mitochondrial dysfunction. The ameliorated level of these fatty acids reflects the improvement of lipid metabolism due to treatment with P. oceanica extract encapsulated in gelatin nanoparticles. Glucosamine is a naturally occurring amino monosaccharide and well known as a precursor for glycosaminoglycans and mucopolysaccharides. The elevations of glucosamine level in the current study are in agreement with Omori et al.,55 who reported that an increase in hexosamines may play a role in hyperglycemia-induced damage of vascular endothelial cells, and thus may contribute to the development of insulin resistance and hypertension. Thus, these findings confirm the ability of P. oceanica extract encapsulated in gelatin nanoparticles as a potent antidiabetic agent.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra09606g |
This journal is © The Royal Society of Chemistry 2021 |