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Phenolics of Moringa oleifera leaves

a b
Lawrence Onyango Arot Manguro & Peter Lemmen
a
Department of Chemistry, Maseno University, P.O. Box 333,
Maseno, Kenya
b
Institut fuer Organische Chemie und Biochemie, Technische
Universitaet Muenchen, Lehrstuhl 1, Lichtenbergstrasse 4, 85747-
Garching, Germany

To cite this article: Lawrence Onyango Arot Manguro & Peter Lemmen (2007) Phenolics of Moringa
oleifera leaves, Natural Product Research: Formerly Natural Product Letters, 21:1, 56-68, DOI:
10.1080/14786410601035811

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 Natural Product Research, Vol. 21, No. 1, January 2007, 56–68

Phenolics of Moringa oleifera leaves

LAWRENCE ONYANGO AROT MANGURO*y and PETER LEMMENz

yDepartment of Chemistry, Maseno University, P.O. Box 333, Maseno, Kenya

zInstitut fuer Organische Chemie und Biochemie, Technische Universitaet Muenchen,
Lehrstuhl 1, Lichtenbergstrasse 4, 85747-Garching, Germany
Downloaded by [Purdue University] at 05:18 22 September 2013

(Received 6 July 2006; in final form 28 September 2006)

Five flavonol glycosides characterised as kaempferide 3-O-(200 ,300 -diacetylglucoside),

kaempferide 3-O-(200 -O-galloylrhamnoside), kaempferide 3-O-(200 -O-galloylrutinoside)-7-O-
-rhamnoside, kaempferol 3-O-[-glucosyl-(1 ! 2)]-[-rhamnosyl-(1 ! 6)]--glucoside-7-O-
-rhamnoside and kaempferol 3-O-[-rhamnosyl-(1 ! 2)]-[-rhamnosyl-(1 ! 4)]--glucoside-
7-O--rhamnoside together with benzoic acid 4-O--glucoside, benzoic acid 4-O--rhamnosyl-
(1 ! 2)--glucoside and benzaldehyde 4-O--glucoside have been isolated from methanolic
extract of Moringa oleifera leaves. Also obtained from the same extract were known
compounds, kaempferol 3-O--rhamnoside, kaempferol, syringic acid, gallic acid, rutin and
quercetin 3-O--glucoside. Their structures were determined using spectroscopic methods
as well as comparison with data from known compounds.

Keywords: Moringa oleifera; Moringaceae; Flavonol glycosides; Benzoic acid glycosides;

Benzaldehyde 4-O--glucoside; Leaves

1. Introduction

Moringa species namely, Moringa oleifera Lam and Moringa stenopetala (Moringaceae)
are plants of considerable economic potential because of their leaves which are used as
vegetables by various communities in the arid and semi-arid regions of Kenya [1,2].
The leaves are also important articles of commerce in local markets [3]. Apart from
being sources of vegetables, the trees also find medicinal applications among various
communities as a remedy for diabetes, typhoid, malaria, hypertension, stomach
problems and amoebic dysentry [3–5]. Elsewhere, pharmacological profiles of
M. oleifera as antibacterial have been reported [6,7]. Both plant seeds are used in the
indigenous system as water purifiers [8–11]. Phytochemical studies on both plants are
few and a chemical evaluation of M. stenopetala leaves [12] led to the isolation of
phenolics, mainly flavonoids and glycosidated benzaldehyde derivatives. In the present
work, the defatted aqueous methanolic extract of M. oleifera leaves was subjected to
phytochemical investigation leading to the isolation of five flavonol glycosides (1–5),
two benzoic acid glycosides (6 and 7) and benzaldehyde 4-O--glucoside (8).
Together with these were known compounds; kaempferol 3-O--rhamnoside (9),

*Corresponding author. Email: kamanguro@yahoo.com

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online ß 2007 Taylor & Francis
http://www.tandf.co.uk/journals
DOI: 10.1080/14786410601035811
 Phenolics of M. oleifera leaves 57

quercetin 3-O--glucoside (10), gallic acid (11), syringic acid (12), kaempferol (13) and
rutin (14) [12,13]. Their structures were established by spectroscopic means and also by
comparison with closely related compounds.
3′
R3
1 4′
1′
R2 O
7

5 4 R1
OH O

1 R1 = O-2′′,3′′-Diacetylglucoside, R2 = OH, R3 = OCH3

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2 R1 = O-[Glucosyl-(1′′′ → 2′′ )]-[rhamnosyl (1′′′′ → 6′′ )-glucoside,

R2 = O-rhamnose, R3 = OH

3 R1 = O-(2′′-Galloylrhamnoside), R2 = OH, R3 = OCH3

4 R1 = O-(2′′-Galloylrutinoside), R2 = O-Rhamnose, R3 = OCH3

5 R1 = O-[Rhamnosyl-(1′′′ → 2′′ )]-[rhamnosyl (1′′′′ → 4′′ )-glucoside,

R2 = O-Rhamnose, R3 = OH

1 4
R4 R5

6 R4 = CO2H, R5 = O-Glucose

7 R4 = CO2H, R5 = O-Rhamnosyl-(1′′ → 2′)-glucose

8 R4 = CHO, R5 = O-Glucose

2. Results and discussion

Compound 1, a yellow amorphous powder (from MeOH–H2O, 99 : 1) afforded a

HRMS molecular ion peak at m/z 546.13734 [M]þ (Calcd m/z 546.12952), consistent
with the C26H26O13 formula. The UV spectrum in MeOH and with diagnostic
customary shift reagents (AlCl3, ACl3/HCl, NaOMe, NaOAc, NaOAc/H3BO3) were in
accordance with a C-3 and C-40 -disubstituted flavonol skeleton [14]. Acid hydrolysis
gave kaempferide and glucose confirmed by TLC and PC co-chromatography with
authentic samples. The large coupling constants of the anomeric proton (d, J ¼ 7.6 Hz)
indicated that glucose unit was present in the -configuration. Accordingly, the
13
C NMR spectrum (table 1) displayed 26 signals which were resolved into three
methyls, one methylene, eleven methines and eleven quaternary carbon atoms by DEPT.
The 500 MHz 1H NMR spectra confirmed the suggestions and, in addition, revealed the
presence of two acetyl groups as evidenced by two singlets at  2.03 and 2.01.
 58 L. O. A. Manguro and P. Lemmen
13
Table 1. C NMR of compounds 1–5 and 9.

Carbon 1 2 3 4 5 9
2 157.1 156.6 156.9 156.6 156.5 157.7
3 134.0 133.4 135.0 133.8 134.6 134.1
4 179.4 177.9 178.2 177.6 178.8 179.4
5 162.0 161.1 161.3 160.8 162.4 162.2
6 99.8 98.9 98.8 99.6 99.6 99.8
7 164.6 162.8 163.1 163.6 163.4 163.5
8 95.0 94.0 94.5 94.6 94.5 95.3
9 158.2 157.2 156.1 157.3 157.9 158.0
10 105.6 105.3 105.1 105.6 105.3 104.6
10 121.4 121.1 121.4 119.8 120.6 122.3
20 130.8 131.5 130.3 130.8 131.5 132.2
30 115.3 116.6 115.0 115.1 115.8 116.8
40 163.2 160.4 163.1 162.9 161.0 160.3
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50 116.3 116.6 115.0 115.1 115.8 116.8

60 130.8 131.5 130.3 130.6 131.5 132.2
100 105.1 104.9 101.5 103.3 104.8 102.7
200 79.0 82.9 73.0 81.8 81.0 71.4
300 81.8 76.0 67.0 77.5 76.1 72.0
400 72.8 70.4 74.5 70.9 80.2 73.1
500 77.0 77.1 69.1 77.7 76.7 69.8
600 62.6 68.6 17.9 67.7 62.3 17.8
1000 103.1 119.7 120.3 101.0
2000 70.2 108.0 108.6 72.0
3000 68.0 147.3 146.0 71.4
4000 72.9 140.1 139.1 72.8
5000 70.3 147.3 146.0 69.5
6000 17.8 108.0 108.6 17.4
10000 101.6 99.4 100.0
20000 73.9 72.0 72.4
30000 76.8 70.4 69.9
40000 70.4 73.2 73.1
50000 77.9 71.1 70.0
60000 61.4 18.0 17.5
100000 98.9 100.3 99.5
200000 69.9 70.0 71.0
300000 68.8 70.2 70.2
400000 71.5 71.8 72.0
500000 69.3 69.4 69.9
600000 17.9 17.9 17.9
CO 168.4 169.0
40 -OMe 56.5 57.4 56.9
OAc 170.3
171.0
23.4
24.1

The positions of the methoxy group as well as the glucose on the aglycone were
established from HMBC experiments (figure 1) which indicated two or three bond H,
C-correlations and in this way, it was proved that the glucose unit was attached
glycosidically to C-3 and the methoxy group at C-40 . Similarly, the positions of
attachment of the acetyl groups were confirmed by the HMBC correlations and by
decoupling experiments whereby H-200 was found to couple with H-300 . On the
basis of spectroscopic data, compound 1 was characterized as kaempferide
3-O-20 ,30 -diacetylglucoside.
 Phenolics of M. oleifera leaves 59

H
OMe

HO O
H
OAc 4′′
AcO
O O OH
1′′ OH
OH O 5′′
H

OH
H

Me O O O
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HO 1′′′′′
H
HO 1′′ H
OH OH 1′′′′
O O O
O O CH3
OH O 1′′′ OH
O HO
H H
OH OH
OH H
HO

HO
HO 2

OMe 6′′′′
O Me 4′′′′
5′′′′′
HO Me O H 1′′′′ OH
OH
O
H HO
4′′′′′ O H 1′′′′
HO 1′′ 5′′ O
1′′′′′ H
O O OH
HO O
OH O 7′′′ CO 2′′ OH
1′′′ H H

HO OH
4′′′
4 OH

HMBC correlations

NOESY correlations

Figure 1. Pertinent HMBC and NOESY correlations for compounds 1, 2 and 4.

Compound 2 exhibited UV data typical of 3,7-O-disubstituted flavonol derivative

[14] and on acid hydrolysis yielded kaempferol, rhamnose and glucose identified by
TLC and PC co-chromatography after comparison with authentic samples.
The FABMS molecular ion peak at m/z 903 [M þ H]þ corresponding to protonated
C39H50O24 in combination with other significant fragments at m/z 757 [M þ H-146]þ,
595 [M þ H-146–162]þ, 433 [M þ H-2
146–162]þ and 287 [M þ H-2
146–2
162]þ
were in accord with kaempferol nucleus containing two glucosyl and two rhamnosyl
moieties. The fragmentation pattern supported by the 1H NMR anomeric peaks at
 4.70 (d, J ¼ 7.4 Hz) and 4.60 (d, J ¼ 1.8 Hz) suggested that one glucosyl and a
rhamnosyl moiety were present as terminal sugars [15,16]. The primary glucose
exhibiting anomeric proton at  5.40 was linked to the aglycone at C-3 as was
 60 L. O. A. Manguro and P. Lemmen

ascertained by the HMBC correlation between the peaks at  5.40 (H-100 ) and  133.4
(C-3), while the rhamnosyl moiety with anomeric at  5.25 was linked at C-7, confirmed
by HMBC correlations between H-100000 ( 5.25) and (C-7)  162.8. Similarly, the HMBC
correlations between H-200 ( 3.74) with the anomeric carbon at  103.1 (C-1000 ) and
CH2-600 ( 3.69 and 3.64) with another anomeric carbon at  101.6 (C-10000 ) identified the
positions 200 and 600 of primary glucose as glycosidic linkage sites, thus confirming that a
terminal glucose was linked at C-200 , whereas the rhamnosyl was at C-600 . The foregoing
evidence was further supported by the 13C NMR signal at  81. 9 attributed to C-200 of
the inner glucose, suggesting a glucosyl-(1 ! 2)-glucosyl moiety (as in sophorosyl)
and the signal at  68.6 attributed to C-600 of inner glucose suggesting a rhamnosyl-
(1 ! 6)-glucosyl moiety (as in rutinosyl) [15,17,18]. The 13C NMR data were in
agreement with [glucosyl-(1 ! 2)]-[rhamnosyl-(1 ! 6)]-glucosyl moiety, as reported for
Downloaded by [Purdue University] at 05:18 22 September 2013

3-substituted kaempferol derivatives [19–21]. Therefore, on the basis of spectroscopic

evidences, compound 2 was formulated as kaempferol 3-O-[-glucosyl-(1 ! 2)]-
[-rhamnosyl-(1 ! 6)]--glucoside-7-O--rhamnoside.
Compound 3, a yellow powder was isolated after repeated flash chromatography
over deactivated silica gel using CH2Cl2–MeOH (9 : 1). It gave a FABMS peak at m/z
599 [M þ H]þ, consistent with the formula C29H26O14, confirmed by 13C NMR
spectrum (29 carbons). The UV spectrum in MeOH showed absorptions at 265
(band II), 312 and 350 nm (band I), suggesting a flavonol skeleton with a sugar residue
linked at C-3 [14]. The bathochromic shift of band I with AlCl3/HCl (50 nm) further
signified a 5-hydroxy-3-O-substituted flavonol; the bathochromic shift of band II
(10 nm) with NaOAc indicated the presence of unsubstituted hydroxyl group at C-7
while the shift of band I (10 nm) with NaOMe suggested the presence of a free
40 -hydroxy group [22,23]. The 1H NMR spectral data confirmed the above features and
in addition, revealed the presence of galloylrhamnoside moiety and a signal for a
methoxy group. Acid hydrolysis yielded kaempferide, rhamnose and gallic acid
identified by TLC and PC co-chromatography with authentic samples. The 13C NMR
spectrum showed similarities with kaempferide 3-O--rhamnoside especially in the
sugar moiety and the benzopyrone ring [22] and the position of the methoxy group was
confirmed by the HMBC correlation between the methoxy group ( 3.70) and C-40
( 163.1). Similarly, the galloyl moiety was assigned to C-200 of the rhamnose on the
basis of correlations between H-200 ( 5.00) with the galloyl C-7000 (C¼O) ( 168.4). Thus,
compound 3 was concluded to be kaempferide 3-O-(200 -O-galloylrhamnoside).
Compound 4 afforded UV data, which suggested a free hydroxyl at C-5 [14].
Acid hydrolysis gave kaempferide, gallic acid, glucose and rhamnose confirmed by TLC
and PC co-chromatography after comparison with authentic samples. The foregoing
evidence was supported by the FABMS spectrum which afforded a molecular ion peak
at m/z 907 [M þ H]þ corresponding to C41H46O23. The positions of substitution of the
galloyl, glucose and the rhamnosyl moieties as well as the complete structure of 4 were
determined by 1H and 13C NMR analysis. The location of the galloyl moiety at C-200
and the rhamnosyl moiety at C-600 , respectively, on the primary glucose were suggested
from the downfield shift of both H-200 ( 4.94) and CH2-600 ( 3.80, 3.65) relative to those
corresponding resonances of kaempferol 3-O-rutinoside [14,24–26]. This structure was
further supported by 1H–13C long-range coupling two dimensional correlated NMR
(figure 1) which showed correlations between glucose H-100 ( 5.35, J ¼ 7.5 Hz) and C-3
( 133.8); primary rhamnosyl H-100000 ( 5.40, J ¼ 1.1 Hz) with C-7 ( 163.6); rhamnose
H-10000 ( 4.44, J ¼ 1.0 Hz) with glucose C-600 ( 67.3), and glucose H-200 ( 3.80) with
 Phenolics of M. oleifera leaves 61

+
OH

Me O O O
HO
HO
OH OH
O O O
O O CH3
O OH
m/z 286 OH HO
Me O OH
HO OH
HO
OH
+
m/z 887 [M + H]

+ OH
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O O O CH3
O
OH HO
HO Me O OH OH
HO m/z 471 [glc + rha + rha + H]+
OH

+
Me O
HO + OH
HO O O O CH3
HO
OH OH
m/z 147 [rha + H]+ HO
OH
OH
m/z 309 [glc + rha + H]+

+ OH
O OH
HO
OH
m/z 163 [Glc + H]+

Figure 2. Fragmentation pattern of compound 5 in FABMS.

galloyl C-7000 (C¼O) ( 169.0). Thus, the complete assignment confirmed compound 4 as
kaempferide 3-O-(200 -O-galloylrutinoside)-7-O--rhamnoside.
Compound 5 showed a molecular ion peak at m/z 887 [M þ H]þ, analyzing for
C39H50O23. Acid hydrolysis afforded rhamnose, glucose and kaempferol identified by
TLC and PC co-chromatography with authentic samples. The aglycone exhibited
1
H NMR data typical of kaempferol moiety [27–30], while UV data recorded in MeOH
and with common shift reagents [14] suggested a kaempferol derivative with free
hydroxyls at C-5 and C-40 , a fact that was corroborated by HMBC correlations. The mass
spectrum fragmentation pattern (figure 2) together with four anomeric proton signals at
 5.21 (d, J ¼ 7.8 Hz),  5.40 (d, J ¼ 1.6 Hz), 4.55 (d, J ¼ 1.2 Hz) and 4.47 (d, J ¼ 1.4 Hz)
indicated the presence of four sugar units in the molecule. Furthermore, a comparative
analysis of the 13C NMR chemical shift of the sugar unit with that of kaempferol
3-O-glucoside [14,24,25] showed glycosylation shift for C-200 (approx. 8.30 ppm) and
 62 L. O. A. Manguro and P. Lemmen

C-400 (approx. 7.0 ppm) in the primary glucose unit, thus suggesting the presence of 200 ,400 -
disubstituted glucose unit bearing two ramnosyls. The 13C NMR signals at  81.0
attributed to C-200 of the primary glucose suggested rhamnosyl-(1 ! 2)-glucose
bioside [31] while the signal at  80.2 attributed to C-400 of the same glucose indicated
rhamnosyl-(1 ! 4)-glycoside moiety [32,33], a fact further suggested by NOESY and
HMBC spectra correlations. Similarly, the other rhamnosyl moiety was assigned to C-7
on the basis of shift of C-7 at  163.4 in comparison with kaempferol 3-O-rutinoside-40 -O-
diglucoside [34], further ascertained by HMBC correlation between H-1000 ( 5.40) and
C-7 ( 163.4). Thus, the 13C NMR data were in good agreement with [rhamnosyl-
(1 ! 2)]-[-rhamnosyl-(1 ! 4)]-glucosyl moiety as described for 3-O-substituted methyl-
protodioscin [12]. Therefore on the basis of spectroscopic evidences, compound 5 was
established as kaempferol 3-O-[-rhamnosyl-(1 ! 2)]-[-rhamnosyl-(1 ! 4)--gluco-
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side-7-O--rhamnoside.
Along with the flavonol glycosides, two benzoic acid glycosides (6 and 7) and
benzaldehyde 4-O--glucoside (8) were isolated after repeated flash chromatography.
Compound 6 was isolated as a light yellow powder. It showed spectral properties of
benzoic acid 4-O-glucoside [12] and on acid hydrolysis yielded p-hydroxybenzoic acid
and glucose identified by TLC and PC co-chromatography with authentic samples.
The p-hydroxybenzoic acid nature was further established from 1H NMR spectrum
data as outlined in experimental section. Similarly, the signal for the anomeric proton of
glucose appeared at  5.15 (d, J ¼ 7.7 Hz) while those for H-20 , H-30 , H-40 and H-50 were
centred at  3.55, 3.46, 3.40 and 3.20, respectively. Signals at  3.65 and 3.50, each
doublet were due to the terminal-CH2OH group in glucose. The assignment of the
1
H NMR was supported by the 13C NMR data which exhibited a total of 13 carbon
atoms sorted out into one methylene, nine methine and three quaternary carbons by
DEPT, a fact further confirmed by the FABMS molecular ion at m/z 301 [M þ H]þ
corresponding to a formula of C13H16O8. The position of the sugar attachment was
established from HMBC cross peaks between H-10 ( 5.15) and C-1 ( 137.3).
Thus, based on spectroscopic evidence, compound 6 was deduced as benzoic acid
4-O--glucoside
Compound 7 was analyzed for C19H26O12 (FABMS, 13C NMR and DEPT). Data
from 1H and 13C NMR indicated a secondary metabolite with structure closely similar
to that of compound 6 except for the rhamnosyl moiety. Acid hydrolysis afforded
p-hydroxybenzoic acid, glucose and rhamnose identified by TLC and PC
co-chromatography with authentic samples. The positions of the rhamnosyl moiety
and the primary glucose on the aglycone were established from HMQC and HMBC
experiments which proved that the glucose unit was attached glycosidically at C-4 while
the rhamnosyl moiety was at C-20 of the glucose molecule, further evidenced by the
13
C NMR downfield shift of glucose C-20 shift at  81.1. Thus, compound 7 was
concluded to be benzoic acid 4-O--rhamnosyl-(1 ! 2)--glucoside.
Compound 8 was light yellow in colour and its IR spectrum showed a significant
absorption peak at 1620 cm1 characteristic of a conjugated aldehyde [12], a fact further
supported by a sharp singlet at  9.96 (CHO) in the 1H NMR spectrum. Other signals
displayed at  7.91 (d, J ¼ 9.0 Hz, H-3 and H-5) and 7.24 (d, J ¼ 9.0 Hz, H-2 and H-6)
were typical of para-substituted benzaldehyde [12]. Acid hydrolysis yielded glucose as
the sugar residue confirmed by TLC and PC co-chromatography with authentic sample.
Both the 1H and 13C NMR of compound 8 were similar to those of 6 with major
structural difference being the aldehyde moiety, fact corroborated by the FABMS
 Phenolics of M. oleifera leaves 63

molecular ion peak at m/z 285 [M þ H]þ, analysing for protonated C13H16O7 and a
13
C NMR peak at  193.4. The positions for the glucosyl and aldehyde moieties
were similarly confirmed by HMBC to be at C-1 and C-4, respectively. Thus, on
the basis of spectroscopic evidence, compound 8 was concluded to be benzaldehyde
4-O--glucoside.

3. Experimental

3.1. General experimental procedures

The UV and IR data were obtained on Hewlett-Packard Array 8452 A spectro-
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photometer and Perkin-Elmer FT-IR 600 series, respectively. FAB mass spectral data
were measured on a VG ZABSPEC. The 1H NMR data were taken in CDCl3þ drop
DMSO-d6 and DMSO-d6 on a Bruker NMR advance Ultrashield TM spectrometer
operating at 500 and 125 MHz. The EIMS data were measured using 70 eV MAT
8200 A Varian Bremen instrument. Preparative HPLC was performed on JASCO
Labor and Daten Technik Deutschland GmBH (RP-18, 250
20 mm, 7.4 mm JASCO
Kromasil 100). Silica gel for column and TLC plates were impregnated with 2% oxalic
acid solution.

3.2. Plant material

Moringa oleifera leaves were collected at Ramogi hill, Kenya Forestry Research
Institute Station on the Bondo-Usenge highway in January 2003. Voucher specimens
were identified after comparison with authentic samples at the Kenya National
Museum Herbarium.

3.3. Extraction and isolation

The CH2Cl2 defatted powdered leaves (approx. 1 kg) were further extracted with
aqueous MeOH (2.5 L
3) for 2 weeks. The extracts were combined and freed from the
solvent under reduced pressure to afford a dark green semi solid residue 85 g. A portion
of the extract 60 g was pre-adsorbed onto silica gel and then chromatographed over
deactivated silica gel with CH2Cl2–MeOH gradient to pure MeOH affording 200
fractions of 50 mL each. The composition of the fractions were monitored by TLC
using eluent CH2Cl2–MeOH (9 : 1, 3 : 2) and those that showed similar profiles were
combined to constitute four major pools (I–IV). Pool 1 (fractions 4–35, 11.0 g) was
further repeatedly purified by flash chromatography using eluent CH2Cl2–MeOH
(99 : 1) followed by the same solvent system in the ratio 19 : 1 to afford gallic acid
(11, 75 mg), kaempferide 3-O-200 ,300 -diacetylglucoside (1, 45 mg), kaempferol (13, 31 mg)
and syringic acid (12, 52 mg). Fractions 38–92 (pool 11, 10.5 g) was similarly subjected
to repeated flash chromatography using CH2Cl2–MeOH (19 : 1) followed by the same
solvent in the ratio 9 : 1, collecting 20 mL each to give benzoic acid 4-O--glucoside
(6, 50 mg), kaempferol 3-O--rhamnoside (9, 75 mg), benzaldehyde 4-O--glucoside
(8, 40 mg) and quercetin 3-O--glucoside(10, 37 mg).
 64 L. O. A. Manguro and P. Lemmen

Pool III (fractions 93–147, 8.5 g) upon further purification using CH2Cl2–MeOH
(9 : 1) followed by the same solvent in the ratio 4 : 1 and 7 : 3, collecting 10 mL each gave
rutin (14, 95 mg), kaempferide 3-O-(200 -galloylrhamnoside) (3, 45 mg) and benzoic acid
4-O--rhamnosyl-(1 ! 2)--glucoside (7, 83 mg). Pool IV (fractions 148–200, 16 g)
mainly from methanol elution was re-chromatographed using CH2Cl2–MeOH mixture
of increasing polarity of the latter to give 150 fractions of 20 mL each. The eluents
contained three major components which were purified by preparative HPLC reverse
phase using gradient elution of acetonitrile–H2O (starting with 75% acetonitrile and
25% H2O and finishing with 100% H2O; mobile flow rate, 12 mL min–1 and pressure
of 6.1 mPa; time per run was 50 min), injecting 10 mL each time to give kaempferide
3-O-[-glucosyl-(1 ! 2)]-[-rhamnosyl-(1 ! 6)]--glucoside (2, 27 mg), kaempferide
3-O-200 -galloylrutinoside7-O--rhamnoside (4, 24 mg) and kaempferol 3-O-[-
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rhamnosyl(1 ! 2)]-[-rhamnosyl-(1 ! 4)]--glucoside-7-O--rhamnoside (5, 34 mg),

respectively.

Kaempferide 3-O-200 ,300 -diacetylglucoside (1). Amorphous yellow powder from

MeOH–H2O (99 : 1), m.p. 245–247 C, ½25 D 11.5 (c 1.0, MeOH); UV max (MeOH)
266, 316, 352; þAlCl3: 269, 308, 352, 402; þAlCl3/HCl: 270, 308, 350, 398; þNaOMe:
272, 314, 386; þNaOAc: 270, 350, 390; NaOAc/H3BO3: 268, 318, 350, 367 nm; IR max
(KBr) 3500 (OH), 2970, 2825, 1734, 1680, 1650, 1470, 1410, 1220, 1050, 1010, 850 cm–1;
1
H NMR (CDCl3 þ drop DMSO-d6) ppm: 12.60 (s, OH-5, D2O exchangeable), 10.30
(s, OH-7, D2O exchangeable), 7.98 (d, J ¼ 8.7 Hz, H-20 and H-60 ), 6.95 (d, J ¼ 8.7 Hz, H-
30 and H-50 ), 6.32 (d, J ¼ 2.1 Hz, H-8), 6.21 (d, J ¼ 2.1 Hz, H-6), 3.60 (s, 40 -OMe); 3-O-
glucose: 5.20 (d, J ¼ 7.6 Hz, H-100 ), 5.01 (m, H-200 ), 4.90 (m, H-300 ), 3.70 (m, H-600 a), 3.52
(m, H-600 b), 3.40 (m, H-400 ), 3.35 (m, H-500 ), 2.03 (s, OAc), 2.01 (s, OAc); 13C NMR
(CDCl3 þ drop DMSO-d6) data, see table 1; FABMS (positive ion mode) m/z 547
[M þ H]þ (3), 301 [M þ H-diacetylglucosyl]þ (40), 153 (35), 137 (55); HRMS m/z
546.1373 (Calcd for C26H26O13, 546.1295).

Kaempferol-3-O-[b-glucosyl-(1 ! 2)]-[a-rhamnosyl-(1 ! 6) -O-b-glucoside-7-O-a-

rhamnoside (2). A yellow amorphous powder from MeOH–H2O (99 : 1), m.p.
4250 C, ½25 D 37 (c 0.6, MeOH); UV max (MeOH) 265, 310, 358; þAlCl3: 274, 315,
340, 357, 412; þAlCl3/HCl: 271, 315, 354, 410; þNaOMe: 273, 314, 382; þNaOAc: 272,
360, 412; þNaOAc/H3BO3: 272, 314, 360, 412 nm; IR max (KBr) 3430 (OH), 3240,
2940, 2827, 1685, 1640, 1580, 1500, 1210, 1050, 1010, 776 cm–1; 1H NMR (DMSO-d6,
99%) ppm: 12.70 (s, OH-5, D2O exchangeable), 7.76 (d, J ¼ 8.6 Hz, H-20 and H-60 ), 6.96
(d, J ¼ 8.6 Hz, H-30 and H-50 ), 6.31 (d, J ¼ 2.1 Hz, H-8), 6.22 (d, J ¼ 2.1 Hz, H-6); 3-O-
glucose: 5.40 (d, J ¼ 7.6 Hz, H-100 ), 3.74 (m, H-200 ), 3.69 (m, H-600 a), 3.64 (m, H-600 b),
3.57 (m, H-300 ), 3.48 (m, H-500 ), 3.28 (m, H-400 ); 200 -O-glucose: 4.70 (d, J ¼ 7.4 Hz, H-1000 ),
3.65 (m, H-6000 a), 3.56 (m, H-5000 ), 3.52 (m, H-6000 b), 3.47 (m, H-2000 ), 3.94 (m, 3000 ), 3.31
(m, H-4000 ), 600 -O-rhamnose: 4.60 (d, J ¼ 1.8 Hz, H-10000 ), 3.70 (m, H-30000 ), 3.58 (m, H-20000 ),
3.40 (m, H-50000 ), 3.27 (m, H-40000 ), 1.0 (d, J ¼ 6.2 Hz, Me-60000 ); 7-O-rhamnose: 5.25 (d,
J ¼ 1.0 Hz, H-100000 ), 3.62 (m, H-200000 ), 3.54 (m, H-300000 ), 3.46 (m, H-500000 ), 3.29 (m, H-400000 ),
1.02 (d, J ¼ 5.9 Hz, Me-600000 ); 13C NMR (DMSO-d6) data, see table 1; FABMS (positive
ion mode) m/z 903 [M þ H]þ (4), 757 [M þ H-146]þ (8), 595 [M þ H-146–162] þ (11),
471 [H þ glc þ 2rha]þ (12), 433 [M þ H-146–2
162]þ (7), 309 [glc þ rha þ H]þ (5),
 Phenolics of M. oleifera leaves 65

287 [M þ H-2
146–2
162]þ (30), 153 (65), 137 (51); HRMS m/z 902.2692 (Calcd for
C39H50O24, 902.2614).

Kaempferide 3-O-(200 -O-galloylrhamnoside) (3). A yellow amorphous powder from

CH2Cl2–MeOH (9 : 1), m.p. 238–240 C; ½25 D 81 (c 0.5, MeOH); UV max (MeOH)
265, 312, 350; þAlCl3: 272, 308, 350, 400; AlCl3/HCl: 272, 306, 352, 400; þNaOMe:
274, 312, 360; þNaOAc: 275, 352, 392; NaOAc/H3BO3: 268, 316, 360 nm; IR max
(KBr) 3500 (OH), 3230, 2975, 2870, 1730, 1680, 1650, 1570, 1410, 1370, 1225, 1170,
1020, 920, 850 cm–1; 1H NMR (CDCl3 þ DMSO-d6) ppm: 12.75 (s, OH-5, D2O
exchangeable), 10.50 (s, OH-7, D2O exchangeable), 8.00 (d, J ¼ 8.6 Hz, H-20 and H-60 ),
6.96 (d, J ¼ 8.6 Hz, H-30 and H-50 ), 6.28 (d, J ¼ 1.9 Hz, H-8), 6.16 (d, J ¼ 1.9 Hz,
H-6), 3.70 (s, 40 -OMe); 3-O-rhamnose: 5.10 (d, J ¼ 1.2 Hz, H-100 ), 5.00 (m, H-200 ), 3.57
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(m, H-400 ), 3.43 (m, H-500 ), 3.30 (m, H-300 ), 0.90 (d, J ¼ 6.2 Hz, Me-600 ); 200 -O-galloyl: 7.20
(s, H-2000 and H-6000 ); 13C NMR (CDCl3 þ DMSO-d6) data, see table 1; FABMS
(positive ion mode) m/z 599 [M þ H]þ (5), 447 [M þ H-152]þ (11), 301 [M þ H-152–
146]þ (6), 153, 137; HRMS m/z 598.1323 (Calcd for C29H26O14, 598.1224).

Kaempferide 3-O-(200 -O-galloylrutinoside)-7-O-a-rhamnoside (4). A yellow powder

from MeOH–H2O (99 : 1), m.p. 4250 C, ½25 D 46.5 (c 0.5, MeOH); UV max
(MeOH) 267, 312, 352; þAlCl3: 270, 310, 351, 400; þAlCl3/HCl: 274, 308, 402;
þNaOMe: 272, 312, 386; þNaOAc: 270, 310, 3352; þNaOAc/H3BO3: 270, 310, 352 nm;
IR max (KBr) 3550–3340 (OH), 3280, 2980, 2955, 2850, 1700 (ester), 1680
(,-unsaturated CO), 1600 (C¼C), 1020–1010 (C–O) cm–1; 1H NMR (DMSO-d6,
99%) ppm: 12.65 (s, OH-5, D2O exchangeable), 8.01 (d, J ¼ 8.8 Hz, H-20 and H-60 ), 7.20
(d, J ¼ 8.8 Hz, H-30 and H-50 ), 6.34 (d, J ¼ 2.0 Hz, H-8), 6.26 (d, J ¼ 2.0 Hz, H-6), 3.70
(s, 40 -OMe); 3-O-glucose: 5.35 (d, J ¼ 7.5 Hz, H-100 ), 4.94 (m, H-200 ), 3.80 (m, H-600 a),
3.65 (m, 600 b), 3.50 (m, H-300 ), 3.38 (m, H-500 ), 3.27 (m, H-400 ); 200 -O-galloyl: 7.00 (s, H-2000
and H-6000 ); 600 -O-rhamnose: 4.44 (d, J¼1.0 Hz, H-10000 ), 3.48 (m, H-30000 ), 3.39 (m, H-50000 ),
3.34 (m, H- 20000 ), 3.18 (m, H-40000 ), 1.05 (d, J ¼ 6.3 Hz, Me-60000 ); 7-O-rhamnose: 5.40
(d, J ¼ 1.1 Hz, H-100000 ), 3.70 (m, H-200000 ), 3.61 (m, H-300000 ), 3.51 (m, H-500000 ), 3.35
(m, H-400000 ), 0.98 (d, J ¼ 6.2 Hz, Me-600000 ); 13C NMR (DMSO-d6) data, see table 1;
FABMS m/z 907 [M þ H]þ (4), 761 [M þ H-146]þ (10), 755 [M þ H-152]þ (7), 615
[M þ H-2
146]þ (9), 609 [M þ H-152–146]þ (5), 463 [M þ H-152–2
146]þ (38), 309
[rha þ glc þ H]þ (11), 269 (10), 153 (18), 136 (37); HRMS m/z 906.2430 (Calcd for
C40H46O23, 906.2352).

Kaempferol-3-O-[a-rhamnosyl-(1 ! 2)]-[a-rhamnosyl-(1 ! 4)-O-b-glucoside-7-O-a-rham-

noside (5). A yellow powder from MeOH–H2O (99 : 1), m.p. 4250 C, ½25 D 18.5
(c 1.0, MeOH); UV max (MeOH) 268, 312, 358; þAlCl3: 272, 314, 413; þAlCl3/HCl:
271, 315, 337, 410; þNaOMe: 272, 298, 360, 411; þNaOAc: 264, 360, 414; þNaOAc/
H3BO3: 264, 360, 414 nm; IR max (KBr) 3380 (OH), 3240, 2950, 2822, 1680, 1420, 1380,
1170, 1010, 1010, 770 cm–1; 1H NMR (DMSO-d6, 99%) ppm: 12.70 (s, OH-5, D2O
exchangeable), 8.50 (OH-7, D2O exchangeable), 7.98 (d, J ¼ 8.9 Hz, H-20 and H-60 ), 7.01
(d, J ¼ 8.9 Hz, H-30 and H-50 ), 6.40 (d, J ¼ 2.0 Hz, H-8), 6.28 (d, J ¼ 2.0 Hz, H-6); 3-O-
glucose: 5.21 (d, J ¼ 7.8 Hz, H-100 ), 3.80 (m, H-200 ), 3.64 (m, H-600 a), 3.62 (m, H-300 ), 3.50
(m, H-600 b), 3.43 (m, H-500 ), 3.33 (m, H-400 ); 200 -O-rhamnose: 4.55 (d, J ¼ 1.2 Hz, H-1000 ),
3.75 (m, H-2000 ), 3.41 (m, H-3000 ), 3.35 (m, H-4000 ), 3.27 (m, H-5000 ), 0.95 (d, J ¼ 6.5 Hz,
 66 L. O. A. Manguro and P. Lemmen

Me-6000 ); 400 -O-rhamnose: 4.47 (d, J ¼ 1.4 Hz, H-10000 ), 3.81 (m, H-20000 ), 3.60 (m, H-30000 ),
3.45 (m, H-50000 ), 3.34 (m, H-40000 ), 1.00 (d, J ¼ 6.1 Hz, Me-60000 ); 7-O-rhamnose: 5.40 (d,
J ¼ 1.6 Hz, H-100000 ), 3.76 (m, H-200000 ), 3.59 (m, H-300000 ), 3.49 (m, H-500000 ), 3.31 (m, H-400000 ),
1.16 (d, J ¼ 6.4 Hz, Me-600000 ); 13C NMR (DMSO-d6) data, see table 1; FABMS (positive
ion mode) m/z 887 [M þ H]þ (1), 741 [M þ H-146]þ (5), 595 [M þ H-2
146] þ (2), 499
[M þ H-2
146–162]þ (9), 455 [glc þ 2rha þ H]þ (5), 433 (14), 309 [glc þ rha þ H]þ (8),
287 [M þ H-3
146–162]þ (21), 269 (13), 163 (16), 153 (45), 147 (4), 137 (61); HRMS
m/z 886.2743 (Calcd for C39H50O23, 886.2665).

Benzoic acid 4-O-b-glucoside (6). Light yellow powder from CH2Cl2–MeOH (9 : 1),
m.p. 225–227 C, ½25 D 98 (c 0.5, MeOH); UV max (MeOH) 227, 280, 380 nm; IR max
(KBr) 3450 (OH), 2925, 2850, 1640, 1618, 1510, 1342, 1070, 1020, 980, 830, 770 cm–1;
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1
H NMR (CDCl3 þ drop DMSO-d6) ppm: 7.90 (d, J ¼ 8.4 Hz, H-2 and H-6), 7.20
(d, J ¼ 8.4 Hz, H-3 and H-5); 4-O-glucose: 5.15 (d, J ¼ 7.7 Hz, H-10 ), 3.65 (m, H-60 a),
3.55 (m, H-20 ), 3.50 (m, H-60 b), 3.46 (m, H-30 ), 3.40 (m, H-40 ), 3.20 (m, H-50 ); 13C NMR
(CDCl3 þ drop DMSO-d6) ppm: 131.0 (C-1), 130.0 (C-2), 129.5 (C-3), 137.3 (C-4), 129.5
(C-5), 130.0 (C-6), 168.1 (C-7), 103.6 (C-10 ), 74.8 (C-20 ), 78.0 (C-30 ), 70.7 (C-40 ), 75.0
(C-50 ), 61.2 (C-60 ); FABMS (positive ion mode) m/z 301 [M þ H]þ (4), 162 (glc) (4),
139 [M þ H-162]þ (17), 95 (6) 41 (38); HRMS m/z 300.0845 (Calcd for C13H16O8,
300.0767).

Benzoic acid 4-O-a-rhamnosyl-(1 ! 2)-b-glucoside (7). Pale yellow amorphous powder

from CH2Cl2–MeOH (9 : 1), m.p. 4250 C, ½25 D 61 (c 0.2, MeOH); UV max (MeOH)
230, 266, 357 nm; IR max (KBr) 3430 (OH), 3250, 2930, 2845, 1640, 1514, 1230, 1030,
1010, 776 cm–1; 1H NMR (CDCl3 þ drop DMSO-d6) ppm: 7.80 (d, J ¼ 8.8 Hz, H-2 and
H-6), 7.30 (d, J ¼ 8.8 Hz, H-3 and H-5); 4-O-glucose: 4.90 (d, J ¼ 7.8 Hz, H-10 ), 3.78 (m,
H-20 ), 3.55 (m, H-60 a), 3.47 (m, H-60 b), 3.38 (m, H-30 ), 3.29 (m, H-40 ), 3.24 (m, H-50 );
20 -O-rhamnose: 4.40 (d, J ¼ 1.4 Hz, H-100 ), 3.70 (m, H-200 ), 3.61 (m, H- 500 ), 3.44
(m, H-300 ), 3.23 (m, H-400 ), 0.94 (d, J ¼ 6.5 Hz, Me-600 ); 13C NMR (CDCl3 þ drop
DMSO-d6) ppm: 130.8 (C-1), 129.9 (C-2), 129.2 (C-3), 138.5 (C-4), 129.2 (C-5), 129.9
(C-6), 167.9 (C-7), 102.5 (C-10 ), 81.1 (C-20 ), 78.8 (C-30 ), 71.0 (C-40 ), 74.6 (C-50 ), 62.0
(C-60 ), 99.5 (C-100 ), 69.9 (C-200 ), 68.9 (C-300 ), 72.8 (C-400 ), 68.7 (C-500 ), 17.8 (C-600 );
FABMS (positive ion mode) m/z 447 [M þ H]þ (3), 301 [M þ H-146]þ (6), 309
[glc þ rha þ H]þ (7), 139 [M þ H-146–162]þ (27), 95 (80), 77 (10), 43 (46), 41 (90);
HRMS m/z 446.1424 (Calcd for C19H26O12, 446.1346).

Benzaldehyde 4-O-b-glucoside (8). A light yellow powder from CH2Cl2–MeOH (9 : 1),

m.p. 191–192 C, ½25
D 89 (c 0.6, MeOH); UV max (MeOH) 238, 263, 282 nm; IR max
(KBr) 3450 (OH), 3250, 2925, 2852, 1617, 1490, 1460, 1357, 1050, 860 cm–1; 1H NMR
(CDCl3 þ drop DMSO-d6) ppm: 9.96 (s, CHO), 7.91 (d, J ¼ 9.0 Hz, H-2 and H-6), 7.24
(d, J ¼ 9.0 Hz, H-3 and H-5); 4-O-glucose: 5.10 (d, J ¼ 7.6 Hz, H-10 ), 3.74 (m, H-60 ), 3.60
(m, H-60 a), 3.43 (m, H-30 ), 3.35 (m, H-20 ), 3.24 (m, H-40 ); 3.17 (m, H-50 ); 13C NMR
(CDCl3 þ drop DMSO-d6) ppm: 131.4 (C-1), 129.6 (C-2), 128.8 (C-3), 138.1 (C-4), 128.8
(C-5), 129.6 (C-5), 193.4 (C-7), 101.1 (C-10 ), 74.5 (C-20 ), 79.0 (C-30 ), 70.5 (C-40 ),
74.3 (C-50 ), 62.3 (C-60 ); FABMS (positive ion mode) m/z 287 [M þ H]þ (2), 123
 Phenolics of M. oleifera leaves 67

[M þ H-162]þ (40), 91 (10), 43 (80); HRMS m/z 284.0896 (Calcd for C13H16O7,
284.0818).

3.4. Acid hydrolysis

Compounds 1–8, each 15 mg in a mixture of 8% HCl (2 mL) and MeOH (20 mL) were
separately heated under reflux for 2 h. The reaction mixtures were reduced under
pressure to dryness, dissolved in H2O (3 mL) and neutralized with NaOH. The
neutralized products were then subjected to TLC (eluent: EtOAc–MeOH–H2O–HOAc,
6 : 2 : 1 : 1) and PC (eluent: n-BuOH–HOAc–H2O, 4 : 1 : 5). The sugar chromatograms
were sprayed with aniline hydrogen phthalate followed by heating at 100 C and
were identified after comparison with authentic samples. Similarly, the aglycones were
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confirmed upon exposure to conc. ammonia solution.

Acknowledgements

The authors are grateful to Alexander von Humboldt Stiftung and DAAD for awarding
L. Manguro a fellowship.

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