Antioxidant and hyaluronidase inhibitory activities of diverse phenolics in Phyllanthus emblica

Abstract Fifty-eight phenolic compounds isolated from Phyllanthus emblica were screened and compared for their in vitro and in vivo antioxidant properties, as well as hyaluronidase (HAase) inhibitory activities. Among them, 20 compounds showed to be promising antioxidants due to the stronger scavenging activity in both DPPH radical and Danio rerio reactive oxygen species assays, while nine compounds were potential HAase inhibitors with 100-fold stronger activities than that of the positive control, DSCG. The structure activity relationship was discussed.

. Various chemical constituents have been reported from the titled plant; (Habib-ur-Rehman et al. 2007;El-Desouky et al. 2008;Lv et al. 2014, in supplementary material), and a wide range of bioactivities of P. emblica were associated with its phenolic constituents (Guo et al. 2013;Gaire & Subedi 2016). However, comprehensive and systemic evaluations, particularly the in vivo antioxidant activity on diverse phenolics from P. emblica, are unavailable. Herein, we report the inhibitory activity on hyaluronidase (HAase) and antioxidant activity in DPPH radical and Danio rerio reactive oxygen species (ROS)-scavenging assays of 58 phenolics from P. emblica. The structure activity relationship of the phenolics was discussed.
According to the structural types, the HAase inhibitory ability of phenolics in P. emblica was ellagitannins > condensed tannins > flavonoids > gallotannins > simple phenolics ( Figure S3(f )). As the major phenolics in P. emblica, ellagitannins 1-18 showed mostly the strongest activity. All gallotannins (19-25) also showed stronger activities than the positive control, except 24 with the fewest free hydroxyl group in molecule. Among the simple phenolics 28-33 and 35, only 28 displayed stronger activity (IC 50 = 0.61 mM) than positive control. All the condensed tannins 36-44 and flavonoids 45, 47-54, and 57 showed stronger activities than positive control. Among which, 45 showed the strongest activity (IC 50 = 0.09 mM). The aforementioned results suggested that the HAase inhibition assay showed a clear correlative relationships ( Figure S3(g-h)) between the phenol, especially ortho-dihydroxyphenol numbers. For example, the activity order of 6 > 3 > 9 > 13 was accordant with the phenol and ortho-dihydroxyphenol numbers. The one with more ortho-dihyroxyphenol units exhibit stronger activity. On the other hand, more free carboxyls in molecules decreased the inhibition effects on HAase, e.g. compounds 1 and 25-35.

DPPH radical-scavenging activity of phenolics from P. emblica
The DPPH radical-scavenging activities of 1-58 were shown in Table S1 and Figure S4(a-e). The ellagitannins exhibited the strongest activity. For example, 1-4 showed roughly six-fold stronger activities than positive control. Gallotannins (19-25) and condensed tannins (36-44) also displayed stronger activities than positive control. Of them, the gallotannins (19-21) with more free hydroxyl groups showed the strongest activities. All simple phenolics 25-35 showed the lowest activities, due to less or no ortho-dihydroxyphenol unit in molecules. Flavonoids (45-58) displayed the same radical-scavenging ability with positive control. Among them, 45-47 with galloyl group showed the strongest activities. The above results indicated a clear correlative relationship in DPPH radical-scavenging assay ( Figure S4(g-h)). For example, the less phenol and ortho-dihydroxyphenol numbers (8 < 7 < 1 < 6), the weaker DPPH radical-scavenging ability it have. This was consistent with that reported in the literatures (Wang et al. 2007), indicating that the antioxidant activity of phenolics decreased when less numbers of ortho-dihydroxyphenol units existed in molecules.

Danio rerio ROS-scavenging activity of phenolics from P. emblica
All of the isolates 1-58 were tested for their individual antioxidant activities in zebrafish model (Table S1 and Figure S5(a-e)). Most tested phenolics displayed potential activity in ROS-scavenging assay. At a concentration of 30 μM, 15 compounds with an activity order of 46 > 8 > 41 > 56 > 44 > 42 > 51 > 53 > 2 > 49 > 55 > 58 > 45 > 57 > 35 showed more than 100% ROS clearance rates. Ten of them (44-46, 49, 51, 53, 55-58) were flavonoids, which showed weaker activities than ellagitannins in DPPH radical assay. Most ellagitannins and gallotannins with promising DPPH radical-scavenging activities showed no activity in the ROS-scavenging assay, except ellagitannins 2 and 8. The order of the ROS clearant ability of phenolics in P. emblica was flavonoids > simple phenolics > condensed tannins > gallotannins > ellagitannins ( Figure S5(f )). The aforementioned observation showed that, unlike DPPH radical-scavenging activity, the in vivo antioxidant activity of phenolic compounds has no direct relationship with their free phenol or ortho-dihydroxyphenol units. However, ellagitannins with less free carboxyl groups showed stronger ROS-scavenging abilities. This is consisted with DPPH radical-scavenging activity. Moreover, ellagitannins 2 and 8 with an elaeocarpusinosyl (Ela) ester group, showed the strongest ROS-scavenging activity, indicating that the Ela fragment should be a good scaffold for the in vivo ROS-scavenging activity. The condensed tannins with ortho-trihydroxyphenol exhibited stronger ROS-scavenging activities than the ones with ortho-dihydroxyphenol, and the dimers 37-38 and 40 exhibited weaker activity than the monomers (36, 42).

Conclusion
Most of the phenolics in P. emblica showed potential antioxidative and HAase inhibitory activities. Six ellagitannins (2-3, 7-8, 13, 17), one gallotannin (19), two simple phenolics (31, 33), four condensed tannins (36, 41-42, 44), and six flavonoids (45-46, 49, 51, 53, 55, 58) are promising antioxidants, due to their stronger activities in both DPPH radical and in vivo ROS-scavenging assays. Seven ellagitannins (2, 4-6, 10, 14-15), one gallotannin (22) and one flavonoid (45) were potential HAase inhibitors, due to their 100-fold stronger activities than positive control. The numbers of free phenols and ortho-dihydroxyphenols in molecules increased the activity of phenolics in both HAase and DPPH assays. It is also noted that the existence of more free carboxyl groups in phenolics would impair their antioxidant activities in vivo and in vitro. With the existence of Ela ester group, the phenolics displayed stronger antioxidant potential in vivo ROS-scavenging assay, suggesting Ela unit should be a good scaffold for antioxidative properties. The present study supported that P. emblica represents a valuable natural source of antioxidants and HAase inhibitors with useful potential for food, cosmetic and pharmaceutical industries.