Extraction and Isolation of Kaempferol Glycosides from the Leaves and Twigs of Lindera neesiana

The hydroalcoholic extract of leaves and twigs of Lindera neesiana (Wall. ex Nees) Kurz (Lauraceae) was subjected to various column chromatographic methods that isolated five kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1→2)-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside7-O-α-rhamnopyranoside (1); kaempferol 3-O-β-glucopyranosyl-(1→2)-[α-rhamnopyranosyl (1→6)]β-glucopyranoside (2); kaempferol 3-O-β-glucopyranosyl(1→2)-α-rhamnopyranoside-7-O-αrhamnopyranoside (3); kaempferol 3-O-sophoroside (4); and kaempferol 3-O-α-rhamnopyranoside (5). The extract showed moderate free radical scavenging activity and potent pancreatic lipase inhibitory activity.

Lindera neesiana (Wall.ex Nees) Kurz commonly known as 'Siltimur' in Nepal, is a small tree distributed in Nepal, India, Bhutan, and Myanmar [2,17].The fruits are aromatic and are used as a spice in preparation of various food products as well as traditional medicines to treat diarrhea, tooth pain, headache, and gastric disorders [2,17,18].Essential oils from L. neesiana fruit consisted mainly of Z-citral, E-citral, eucalyptol, citronellal, α pinene and β pinene, and showed potent antibacterial and antifungal effects [19].The water extract from these fruits also showed potent neuroprotective and anti-neuroinflammatory activity [18].However, there was no study on the chemical constituents of the leaves and twigs.In a previous study, we isolated several flavonoids from the leaves and twigs of Lindera sericea, which is widely used as a herbal tea in Japan, and evaluated its free radical scavenging and enzyme inhibitory activities [20].Thus, in this study, we aimed for the isolation and identification of chemical constituents and the evaluation of free radical scavenging and pancreatic lipase inhibitory activities of the extract and compounds.

Plant Materials and Chemicals
The fresh leaves and twigs of Lindera neesiana were collected from Lumle, Kaski District, Nepal on February 2016 and shade dried for two weeks.Voucher specimens were deposited at the Museum of Traditional Medicines, School of Pharmacy, Kumamoto University, Kumamoto, Japan.

Measurement of DPPH Free Radical Scavenging and Pancreartic Lipase Inhibitory Activities
The DPPH free radical-scavenging and pancreatic lipase inhibitory activities were evaluated by using the previously described method [21].

Results
The leaves and twigs of Lindera neesiana (2.3 kg) collected at Lumle, Kaski District, Nepal were extracted with 60% ethanol to obtain 387.0 g of extract.The extract was then subjected to repeated column chromatography on MCI gel CHP20P, Sephadex LH-20, ODS, and silica gel to isolate five pure compounds (1-5) (Figure 1).

Measurement of DPPH Free Radical Scavenging and Pancreartic Lipase Inhibitory Activities
The DPPH free radical-scavenging and pancreatic lipase inhibitory activities were evaluated by using the previously described method [21].
The hydroalcoholic extract and isolated compounds were evaluated for their free radical scavenging activity by DPPH method.The extract showed moderate free radical scavenging activity but the compounds were either very weak or inactive (Table 2).There is a possibility that the extract contains other active molecules which were not isolated in this study.Further bioassay guided isolation and identification of active compounds should be performed in future.

Conclusions
Five kaempferol glycosides were isolated and identified from the hydroalcoholic extract of the leaves and twigs of Lindera neesiana.The extract showed potent pancreatic lipase inhibitory activity.Further studies should focus on the isolation and identification of active compounds and detailed exploration of their activities in vivo.

Figure 1 .Figure 1 .
Figure 1.Photograph of Lindera neesiana (a) and the structures of compounds isolated from the leaves and twigs of Lindera neesiana (b).

Table 2 .
IC 50 values (µg/mL) of extract and compounds for free radical scavenging and lipase inhibitory activities.Values are expressed as the mean ± SD (n = 3), b not active; c Trolox and Cetilistat as positive controls in DPPH free radical scavenging and pancreatic lipase inhibitory activity assays, respectively. a