Antileishmanial and Cytotoxic Compounds from Valeriana wallichii and Identification of a Novel Nepetolactone Derivative

The chloroform extract of Valeriana wallichii (V. wallichii) rhizomes was investigated to elucidate the structures responsible for reported antileishmanial activity. Besides bornyl caffeate (1, already been reported by us previously), bioassay-guided fractionation resulted in two additional cinnamic acid derivatives 2–3 with moderate leishmanicidal activity. The structure of a novel nepetolactone derivative 4 having a cinnamic acid moiety was elucidated by means of spectral analysis. To the best of our knowledge villoside aglycone (5) was isolated from this plant for the first time. The bioassay-guided fractionation yielded two new (compounds 6–7) and two known valtrates (compounds 8–9) with leishmanicidal potential against Leishmania major (L. major) promastigotes. In addition, β-bisabolol (10), α-kessyl alcohol (11), valeranone (12), bornyl isovalerate (13) and linarin-2-O-methylbutyrate (14) were identified. This is the first report on the isolation of 4'-demethylpodophyllotoxin (15), podophyllotoxin (16) and pinoresinol (17) in V. wallichii. In total thirteen known and four new compounds were identified from the extract and their cytotoxic and antileishmanial properties were evaluated.


Bioassay-Guided Fractionation of the Extract
The dried chloroform extract of V. wallichii rhizomes was slurried in MeOH, filtered and fractionated into 12 fractions F1-F12 by preparative HPLC. After screening for antileishmanial activity on L. major promastigotes in vitro the most promising fractions F4-F8 were further investigated by subjecting them to preparative HPLC and multiple column chromatographic separations (cf. Figure 2) which led to the isolation and identification of highly active valtrates 6-9 and cinnamic acid derivatives 1-3 with moderate antileishmanial activity. β-Bisabolol (10) and valeranone (12) were found in a subfraction of F7 and bornyl isovalerate (13) and α-kessyl alcohol (11) were isolated from fraction F8. The non-active fractions F1-F3 gave the highest amount of substances. To avoid missing small amounts of potentially active compounds we have carefully investigated these fractions. This resulted in the discovery of a novel cinnamic acid derivative 4 with a nepetolactone skeleton and villoside aglycone 5. From these fractions 4'-demethylpodophyllotoxin (15), podophyllotoxin (16), pinoresinol (17) and linarin-O-2methylbutyrate (14) were isolated as well (cf. Figure 1).  In the NMR spectra the characteristic 1 H and 13 C signals for a trans-cinnamic acid with a para-substituted phenyl ring were present. 13 C signals in the range of 70-80 ppm represented a sugar moiety. The assignment of the sugar as β-D-glucose was supported by comparison of the coupling constants of the protons to data from the literature [10] while the connectivity of the sugar and the cinnamic acid derivative to the nepetolactone skeleton [11] was established by HMBC cross peaks. Interpretation of coupling constants and NOESY experiments revealed the stereochemistry (cf. V. wallichii filtrated extract (9.5 g) Slurried in MeOH and filtrated over C 18  acid rest and the H-11 protons of the methyl group which led to the conclusion that all these groups are in close spatial proximity. The cinnamic acid seems to be located in the space above H-8. Therefore the proposed configuration is consistent with the configuration of the villoside aglycone 5 and other similar compounds from Valeriana [12]. Additional evidence came from a weak NOESY correlation between the protons of the C-9 methyl group and H-5b. Thus, compound 4 was assigned to be

Structure Elucidation of Villoside Aglycone 5
Villoside is an iridoid glycoside first isolated by Taguchi et al. [13] from Patrinia villosa Juss., a Valerianaceae. However, the villoside aglycone was only obtained semi-synthetically [13]. This is the first report on the isolation of the aglycone 5 directly from a plant, after chromatographic separation of F1 (cf. Figure 2). Similar compounds (jatamanines) have been isolated from V. jatamansi before [12]. Compound 5 was obtained as a yellow syrup. The IR spectrum is characterized by absorption bands at 3387 cm −1 indicating a hydroxyl group and at 1,705 cm −1 caused by a carbonyl function. The ESI-MS showed signals at m/z 185.1 [M+H] + and 207.0 [M+Na] + which was consistent with the calculated mass of m/z 184.1 for the proposed structure. The 1 H spectrum did not show any aromatic proton signals. The 13 C spectrum exhibited ten signals including a typical signal for a lactone functionality at 175.7 ppm. A 13 C signal at 19.8 ppm with a corresponding 1 H signal at 1.13 ppm for three protons suggested the presence of a methyl group (C-11). The methylene groups next to an oxygen gave characteristic 13 C signals at 61.7 and 68.7 ppm (C-3 and C-10, respectively). The remaining signals were found in the aliphatic region. The 1 H-1 H COSY data showed the presence of a spin system involving the hydrogens H-5 to H-9 and, therefore, suggested the presence of a 5-membered ring. All signals have been fully assigned by means of 1 H-1 H COSY, HMBC and HMQC experiments. The configuration was determined by using coupling constants and NOESY data (cf. Figure 4). NOESY correlations between H-11 and H-9, H-5 and H-10, respectively, suggested the methyl group (C-11) and the proton at C-9 as well as the methylene group at C-10 and the proton at C-5 to be located in the same plane of the molecule. The small coupling constant of H-4 and H-3b (3.2 Hz) supports this hypothesis of a quasi-equatorial position of H-4. Additionally, the coupling constant between H-9 and H-8 (Jaa = 8.1 Hz) was backing a transconfiguration. Furthermore, the coupling constant between H-5 and H-9 (11.0 Hz) was consistent with the reported values of the structurally similar jatamanines having the same configuration [12]. Taking biogenetic reasons into account, compound 5 can be assigned to (4R,5S,8R,9S)-4-(hydroxymethyl)-8methylhexahydro-cyclopenta[c]pyranone (villoside aglycone).

Valtrates with Antileishmanial Activity
Typical and well known constituents of Valeriana species are iridoid esters e.g., of the valepotriate and valtrate type, which have been previously investigated in detail because of their in vitro antifungal [14] and cytotoxic activity [15][16][17] and their potential use as antitumor agents [18]. In the active fractions F4-F7 two known valtrates were found, one (compound 8) of the diene-and one (compound 9) of the hydrine-type [16], and two iridoid esters 6, 7 with an as yet unreported substitution pattern. The known valtrates were identified by comparing their spectral data to the literature data (cf. Table 1). Compound 8 has been reported before [14,19,20] and valechlorine (9) too [17,21,22]. The new compounds consist of a typical valtrate skeleton of the hydrine-type and are esterified with various acids e.g. isovaleric acid, acetic acid. The connections of the esters were assigned by HMBC experiments (cf. Figure 5). By comparing coupling constants and taking biogenetic considerations into account the configuration was determined to be the same as reported in the literature.

Antileishmanial Activity and Cytotoxicity
All compounds isolated were subjected to the evaluation of their antileishmanial activity against L. major promastigotes in vitro and cytotoxicity against macrophages (J774.1 murine cell line) using the corresponding AlamarBlue © assays [34] (cf. Table 2). The valtrates 6-9 showed the highest antileishmanial activity (IC50 0.8-2.3 µg/mL). Their cytotoxicity is in the same range. It is unknown whether the cytotoxicity is directly responsible for the leishmanicidal potential. Nevertheless, the valtrates represent the main active principle of the extract. Fractions with moderate antileishmanial activity in the range of the standard drug miltefosine were phenylpropanoids bornyl caffeate (1), bornyl isoferulate (2) and bornyl 3-hydroxycinnamate (3). This has led to the synthesis of a compound library and QSAR studies recently reported by us [2].

Plant Material
Dried rhizomes of V. wallichii were acquired from a local herb shop in Kolkata, India. The identity as V. jatamansi Jones syn. V. wallichii DC. was confirmed by comparison with an authentic sample cultivated at the Institute of Himalayan Bioresource Technology in Palampur, Himachal Pradesh, India by R.D. Singh. A voucher specimen of the herbarium was preserved in the laboratory of Banasri Hazra (IHBT Ref. No. 11666).

Extraction and Isolation
The pulverized rhizome (10 g) was refluxed with chloroform (100 mL) for 2 h and filtered. The solvent was removed from the filtrate in a rotary evaporator, followed by complete drying in vacuo. Thus, the chloroform extract was obtained (yield = 1.1%; w/w) and preserved at 4 °C. The process was repeated to get higher amounts of the extract as per experimental requirements. Dry extract (11 g) was slurried in MeOH and filtrated using a short column filled with LiChroprep RP-18 silica gel which yielded 9.5 g of extract after evaporation. This material was fractionated into 12 fractions (  (15) were identified and F1-7 (2.9 mg) contained pinoresinol (17).

Antileishmanial and Cytotoxicity Assays
Antileishmanial activity against L. major promastigotes and cytotoxicity against a murine macrophage cell line (J774.1) was determined using an AlamarBlue © assay. Methods for both procedures have been reported previously [34]. IC50 values are presented as mean values of two experiments.

Conclusions
The valtrates 6-9 represent the main active antileishmanial principles of the V. wallichii extract, but they are toxic. Additionally, the cinnamic acid derivatives 1-3 showed moderate activity, interestingly except for novel compound 4. This could be due to the larger size of the molecule or the higher polarity caused by the sugar moiety. However, the previously reported cinnamic acid derivatives exhibited higher activity, even without the double bond [2].