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Bio-Active Constituents of Rotenoids Resin

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Comparison between Local Plant Extract and
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 Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

AENSI Journals
Advances in Environmental Biology
ISSN:1995-0756 EISSN: 1998-1066

Journal home page: http://www.aensiweb.com/aeb.html

Bio-Active Constituents of Rotenoids Resin Extracted from Derris elliptica Roots:

Comparison between Local Plant Extract and SAPHYR (France) Cube Resin
1
Saiful Irwan Zubairi, 2Mohamad Roji Sarmidi and 2Ramlan Abdul Aziz
1
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, The National University of Malaysia, 43600 UKM
Bangi, Malaysia
2
Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Skudai, Malaysia

ARTICLE INFO ABSTRACT

Article history: It is well known now that some plant species represent an efficient factory of
Received 14 January 2014 chemicals, which are manufactured and used as bio-weapons against pest attacks.
Received in revised form 19 Extensive work has been done since the last few decades on these potentially useful
April 2014 compounds. During the last few decades a growing interest was directed towards a safer
Accepted 23 April 2014 agricultural production such as free residual toxicity hazards to human beings and to the
Available online 5 May 2014 environment. For instance, plant-based extracts biocides possess a greater advantage
compared with the chemical ones. Their efficacies are also acceptable. In this work, the
aim is to standardize and determine the bio-active constituents extracted from local
Keywords: plant of Derris species (Derris elliptica) by using an internal standard method of
Derris elliptica, Rotenone, Rotenoids, reversed-phase high performance liquid chromatography (RP-HPLC) system. The raw
Cube resin, Internal standard, HPLC plants were collected from Kota Johor Lama, Malaysia and sorted to collect the roots
and stems. Rotenoids resin from the roots and stems were extracted by using a normal
soaking extraction (NSE) method at 30 2 oC. A solvent of acetone with an optimum
solvent-to-solid ratio of 10 ml/g was utilized in the extraction process. The extraction
was carried out for 24 hrs and the extract was filtered to remove any fine debris prior to
the RP-HPLC analysis. The commercially available rotenoids resin cube of SAPHYR
(France) was analyzed to compare and verify the bio-active constituents available with
the extract of local plant species. The RP-HPLC analysis results showed the elicited
distinguishable patterns of the bio-active constituents between the extract of Derris
elliptica and commercial grade of rotenoids cube resin. This extract has a great
potential to be used as insecticidal products. However, the rotenone content is still low
as compared to the commercial grade rotenoids resin. For that reason, a plant tissue
culture is needed to produce a hybrid species between Derris elliptica and Amazonia
species so that high yield of rotenone could be attained.

2014 AENSI Publisher All rights reserved.

To Cite This Article: Saiful Irwan Zubairi, Mohamad Roji Sarmidi and Ramlan Abdul Aziz. Bio-Active Constituents of Rotenoids Resin
Extracted from Derris elliptica Roots: Comparison between Local Plant Extract and SAPHYR (France) Cube Resin. Adv. Environ. Biol.,
8(4), 904-909, 2014

INTRODUCTION

Derris elliptica or Tuba as it is known locally is an insecticidal plant in Malaysia that has been used for
the purpose of bio-pesticide production. Tuba plant is a kind of woody creeper plant and climber. It needs at
least 75% soil moisture content and the surround temperature should be in between 25 to 30 oC to obtain high
content of the rotenone during its development. A calm area with low acidity soil content will enhance the
production of rotenone [1]. Tuba is a member of the Leguminosae and Fabaceae family which comprises 200
genera and 68 species including 21 species of Tephrosia, 12 of Derris, 12 of Lonchocarpus, 10 of Millettia and
several of Mundula [2]. Three species are found in Malaysia, which are Derris elliptica, Derris malaccensis and
Derris uliginosa. Derris is a climbing plant in Southeast Asia and its roots contain rotenone, a strong insecticide
[3]. Derris elliptica and Derris malaccensis contain approximately 4 to 5% (w/w) rotenone while Lonchocarpus
utilis and Lonchocarpus urucu contain 8 to 10% (w/w) rotenone in dry roots [4]. Rotenone has been found to be
used in many applications besides as insecticide. In addition to its effectiveness for both piercing-sucking
insects, such as aphids and red bugs and chewing insects, especially caterpillars upon plants, it makes excellent
dusts for external parasites of animals such as fleas and lice. The side effect of rotenone to aquatic animals is
minimal [5]. The toxic principles all deteriorate rapidly into dihydrorotenone (non-toxic substance) and water
when exposed to sunlight and air; spray and dusts usually lose their effectiveness within a week after application
Corresponding Author: Saiful Irwan Zubairi, School of Chemical Sciences & Food Technology, Faculty of Science &
Technology, The National University of Malaysia, 43600 UKM Bangi, Malaysia.
Phone: +603-89215989; E-mail: saiful-z@ukm.edu.my
905 Saiful Irwan Zubairi et al, 2014
Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

[6]. The outstanding advantages of this group of poisons are that they are harmless to plants (phyto-toxic),
relatively non-toxic to man and act as both contact and stomach poisons to insects [2]. Even though it has
several outstanding properties, there is one problem has not yet been resolved up to now. A variety of its active
ingredients with respect to different kinds of species have been a major problem ever since. In this paper our
goal is to examine the rotenone content in the extract of local Derris species and to compare it with the
commercially available rotenone resin cube extracted from the Amazonian Derris species. The aim of this work
is to also to produce a standard bio-active constituent profile of rotenone extract from Derris plant species
available in Malaysia.

MATERIALS AND METHODS

Plant collection:
Derris elliptica roots were collected in the state of Johor; Kota Johor Lama, Malaysia.

Raw material preparation:

An important aspect of the phytochemical processing is the pre-processing of the herbal material prior to
extraction. The treatment of the herbal material affects the viability of the phytochemical as well as the
extraction yield. The freshly procured Derris roots had to immediately undergo the cleaning process to remove
dirt and soil. The roots were kept and dried in an oven overnight at 30 2 oC and sorted to collect only the roots
and stems. The roots and stems were cut into small pieces using knife mill prior to grinding.

Rotenoids cube resin:

The commercial grade of dried rotenoids cube resin was obtained from SAPHYR (France) with the purity
of 50% (w/w). The sample was believed to be extracted from the Amazonian native species of Lonchocarpus
nicou and Lonchocarpus urucu dried roots. The cube resin was later dissolved in acetonitrile to a concentration
of 0.22 mg/ml prior to the analysis. The molecular structure of essential bio-active constituents available in the
extract and SAPHYR (France) cube resin are shown in Figure 1.

(a) (b)

Fig. 1: Molecular structure of (a) rotenone and (b) deguelin that contains in the liquid crude extract and
SAPHYR (France) cube resin [4,7,8].

Extraction process:
The extraction was carried out by soaking 30 g of dried roots and stems in 300 ml of solvent; acetone, 95%
(v/v) for 24 hrs at 30 2 oC. The liquid crude extract (LCE) was then filtered using Whatman filter paper No. 4
with the aid of Altech filter GAST laboratory diaphragm vacuum pump at 300 mbar.

Analysis of liquid crude extract and rotenoidscube resin:

The liquid crude extract and SAPHYR (France) rotenoids cube resin were subjected to a quantitative
analysis using reverse-phase high performance liquid chromatography (RP-HPLC) to determine the yield of
rotenone and other toxic constituents. The ultra violet (UV) photodiode array (PDA) detection was used at a
wavelength of 294 nm. The analysis of the extract solutions was carried out using an internal standard method
(curcumin, analytical grade, 97% (w/w) - SIGMA-Aldrichas an internal standard solution) [9]. The
operational parameters are shown in Table 1. A C-18 Waters Corp. liquid chromatography stainless steel
column with particle size of 10 m (3.9 mm internal diameter 150 mm length), analytical grade of rotenone
standard with known purity (PESTANAL, analytical grade, 96.2% (w/w); SIGMA-Aldrich), analytical grade
of acetonitrile; 99.9% (v/v) and deionized water (DOW) were utilized in the analysis. The mobile phase system
906 Saiful Irwan Zubairi et al, 2014
Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

was prepared by diluting 3000 ml of acetonitrile into 2000 ml of deionized water (60:40) and filtered through a
cellulose nitrate membrane filter (0.45 m pore size filter) to remove impurities and fine dirt [7].

Table 1: Operational parameters of an isocratic solvent system RP-HPLC.

Column temperature Ambient (30 2 oC)
Flow rate 0.7 ml/min
Wavelength () 294 nm
Injection volume 5 l
Retention times (t):
a) Rotenone 3.55 min - 3.60 min
b) Internal standard 2.87 min - 3.00 min

Preparations of standard solutions:

To prepare an internal standard solution, about 0.035 0.01 g of curcumin standard was dissolved in
acetonitrile, making up to a volume of 50 ml of glass-stoppered conical flask. As for rotenone standard solution,
about 0.0137 0.01 g of rotenone standard was dissolved in acetonitrile, making up to a volume of 50 ml of
glass-stoppered conical flask. Rotenone calibration solution: 10 ml of internal standard solution was added into
10 ml of rotenone standard solution using pipette. The mixture was homogenized using a laboratory shaker to
produce a well dissolved solution.

Sample solution preparation and analysis:

To prepare a sample stock solution, 2 ml of the liquid crude extract (LCE) containing unknown rotenone
concentration was transferred into a 50 ml glass-stoppered conical flask. By using the same pipette used for the
calibration solution, 2 ml of the internal standard solution was mixed and diluted to 100 ml in other volumetric
flask. Meanwhile, to start up the analysis, the prepared mobile phase which is a mixture of acetonitrile and water
(60:40) was allowed to go through into the column overnight until the system is equilibrated (flat baseline). 5 l
of the calibration solution and sample solution was injected into the system. Repetitive injections of both
solutions were carried out to achieve stable responses that agree with 1% of the rotenone peak area (or height) to
the internal standard peak area (or height) ratio. Additionally, the peak area (or height) ratio for the sample
solution must not differ by more than 10% from the peak area (or height) ratio for the calibration solution.
Equation 1 shows the formula of calculating the content (mg) and concentration (mg/ml) of rotenone via internal
standard method.

Ax/[x] = F (AIS/[IS]) (1)

Where:
Ax= Peak area of analyte x; [x] = Concentration of analyte x; F = Response factor; AIS = Peak area of internal
standard and [IS] = Concentration of internal standard.

Statistical analysis:
Data is presented as mean standard deviation (sd) of mean. Statistical comparisons were performed using
Students t-test (PASW version 17.0 IBM Co.). A p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Figure 2, 3 and 4 show chromatogram of SAPHYR (France) rotenoids cube resin and sample solution using
an internal standard (IS) solution of curcumin. The area under the curve of each peak (chromatogram) is
proportional to the concentration of each standard solution used and injected into the column. However, if the
external standard is implemented, the sensitivity of the RP-HPLC detector in identifying the specific bio-active
constituents (rotenone) could generally be compromised with different responses to each standard component
used. For that reason, an internal standard method was carried out to determine the concentration of rotenone in
the liquid crude extract of Derris elliptica and to compare the bio-active constituent profile with the
commercially available rotenoids cube resin manufactured by SAPHYR (France). However, the SAPHYRs
rotenoids cube resin had to be analyzed to verify the availability of rotenone and other constituents as a
certificate of analysis (COA) of the given product was not included and disclosed.

Rotenone calibration solution:

The concentration of rotenone standard comprising theoretical amount of tephrosin and deguelin was
prepared as follows: 0.0137 g/50 ml 96.2% (rotenone purity) = 0.26 mg/ml rotenone, purity tephrosin 1.5%
(w/w): (0.015 0.0137 g)/50 ml = 0.004 mg/ml tephrosin and purity deguelin; 0.5% (w/w) = (0.0005
0.0137 g)/50 ml = 0.00014 mg/ml (deguelin). The assumed approximate purity of tephrosin and deguelin was
907 Saiful Irwan Zubairi et al, 2014
Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

based on the data acquisition of the rotenone standard (Fig. 2). Meanwhile, the prepared internal standard
concentration of curcumin can be calculated based on the observed prominent peak chromatogram (Fig. 2):
0.035 g/50 ml 97% (purity) = 0.68 mg/ml curcumin. As the respective bio-active constituent concentration
was determined, the response factors (F) were calculated with respect to its respective area under the curve (Fig.
2). Equation 1 was used to calculate the F value and is presented in Table 2. All respective peaks show different
profiling responses (e.g., area under the curve and retention time) and can be distinguished accordingly
(p<0.05). Rotenone produced the highest peak area under the curve as compared to tephrosin (p<0.05).
However, deguelin was undetected due to its minute amount in the standard solution and low sensitivity of the
detector used.

Number of peak Name of analyte

3 Acetone
4 Internal Standard (IS)
5 Tephrosin
6 Rotenone
7 Deguelin

Sample solution
Rotenone calibration solution

Fig. 2: Chromatogram of sample solution [rotenone crude extract (x) + internal and standard (IS)] calibration
solution [rotenone standard solution + internal standard solution (IS)].

Table 2: Bio-active constituent profiles of analyzed standard solutions.

#
Calibration solution Concentration (mg/ml) Peak area (mV*s) Response factor (F)
Rotenone standard 0.26 8445.57 230 2.08 0.52
Tephrosin 0.004 1111.83 120 17.86 1.22
Deguelin 0.00014 Undetectable Not determined
Internal standard (IS) 0.68 10583.18 173 Not applicable
#
Results shown are means (sd) of 3 injection/sample (n = 3). () p<0.05 compared to the other compounds.

Table 3: Bio-active constituent profiles of analyzed sample solutions.

# *
Sample solution Concentration (mg/ml) Peak area (mV*s)
Rotenone (x) a
1.95 0.52 9174.95 112
Tephrosin (y) 0.03 0.01 622.95 233
1553.47 120
b
Internal standard (IS) 0.014
a
Rotenone concentration (based on Equation 1): 9174.95/[x] = 2.08 (1553.47/0.0136); [x] = 0.039 mg/ml. Thus, the corrected rotenone
concentration using dilution factor: 0.039 mg/ml (100 ml/2 ml) = 1.95 ml. bConcentration of (IS) in the unknown solution is therefore: C
(IS) = 1.36 mg/100 ml = 0.0136 mg/ml. #Results shown are means (sd) of 3 injection/sample (n = 3). () p<0.05 compared to the other
compounds.

Table 4: Yield of bio-active constituents available inside Derris liquid crude extract.
a b
Sample solution Yield (mg) % Yield (w/w)
Rotenone 140.4 0.02 1.40 0.02
Tephrosin 1.44 0.01 0.014 0.01
2,070 0.05 20.70 0.04
c
Rotenoids resin
7,780 0.03 77.84 0.05
d
Other constituents
a
Yield of rotenone (mg) = C (sample) volume of liquid crude extract (ml). bYield of rotenone in dried roots (%) = Yield of rotenone
(mg)/weight of dried roots 100%. Density of LCE: 0.98 0.02 g/ml. Acetone density: 0.791 g/ml. Density of rotenoids resin: 0.82 - 0.791
= 0.03 g/ml. Total volume of LCE: 70.56 0.5 ml. cTheoretical rotenoids resin available inside the LCE = 0.03 70.56 ml = 2.07 g. d10 g
of dried roots consist of dried bark, stem, lipid and wax (exclude active ingredients and resin).
908 Saiful Irwan Zubairi et al, 2014
Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

Sample solution:
To determine the unknown concentration of rotenone and tephrosin (analyte x and y), 2 ml of internal
standard solution (0.68 mg/ml) was added to another 2 ml of unknown solution. The mixed solution was diluted
to 100 ml in volumetric flask. As predicted, the analysis of the mixture showed in similar retention time
(p>0.05) with diverse peak area under the curves of rotenone, tephrosin and curcumin (p<0.05). The bio-active
constituent profiles of the analyzed sample solutions available inside the LCE are shown in Figure 2 and Table
3. The availability of the bio-active constituents especially rotenone was quite consistent with the findings from
the previous reports [10, 11]. Finally, the yield of all constituents available inside the sample solution (LCE)
was calculated based on the area under the curves obtained (Table 4). The yield of rotenone in dried roots was
slightly higher than the previous reported study (1.14% (w/w)) (p<0.05) [13]. In addition, the sample solution
profile was compared with the commercially available rotenoids cube resin manufactured by SAPHYR (France)
(Fig. 3). Comparable peaks retention time was presented in Figure 4. Both solutions presented the same pattern
and this result confirmed the existence of rotenone in the liquid crude extract [9,12].

Number of peak Name of analyte

3 Tephrosin
4 Rotenone
5 Deguelin

Fig. 3: Chromatogram of SAPHYR (France) rotenoids cube resin (Ccube = 0.22 mg/ml).

Number of peak Name of analyte

3 Acetone
4 Internal Standard (IS)
5 Tephrosin
6 Rotenone
7 Deguelin

SAPHYR (France) solution

Sample solution

Fig. 4: Comparison between SAPHYR (France) rotenoids cube resin and sample solution.
 909 Saiful Irwan Zubairi et al, 2014
Advances in Environmental Biology, 8(4) March 2014, Pages: 904-909

Conclusion:
The results showed that rotenone extracted from local plant species was comparable to the standard
analytical grade purchased from SIGMA-AldrichTM. It was also identical to the commercial grade rotenoids
cube resin given by SAPHYR (France). Based on the chromatogram in Figure 3, besides rotenone (6), the liquid
crude extract consisted of other insecticidal compounds which were tephrosin (5) and deguelin (7). These
compounds are essential for the insecticidal action against the Lepidopteron insect pest of cabbage (Spodoptera
litura). Furthermore, the analysis method used in this work was based on the AOAC official method. However,
several parameters were adjusted in order to achieve high accuracy in identifying bio-active constituents.
Moreover, the internal standard method used was considered the most reliable and accurate analysis method
employed as compared to the external standard method. This method not only has all the advantages of the
external standard method but it also compensates for variations in injection volume and for small changes in
detector sensitivity or chromatographic changes that might have occurred. Because we do not need to inject
exactly the same amount each time, this method generally has better precision than the use of an external
standard. On top of that, ultra clean apparatus, equipment and chemical should also be given due consideration
in order to obtain the optimum result of separation.

ACKNOWLEDGEMENT

The authors wish to acknowledge the kind assistance of the following individual, En. Khairul Annuar Ramli
from Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM). This research was
supported by an IRPA grant 09-02-06-0083 EA261 under the Ministry of Science, Technology and
Environment, Malaysia (MOSTE).

REFERENCES

[1] Grinda, F. and J. Gueyne, 1986. Extraction of insecticides from plants. USPTO PATENT FULL -TEXT
AND IMAGE DATABASE, SAPHYR S.A.R.L. (France) (U.S Patent: 4,698,222).
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insecticide from Tephrosia candida. J. Agri. Food Chem. 40: 1208-1210.
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[6] Schnick, R.A., 1974. Review of the literature on the use of rotenone in fisheries. FWS-LR-74/15, NTIS
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7423:130.
[7] Rodney, B. and H. Alan, 1976. Determination of rotenone in pesticide formulations and the separations of
six rotenoids by reversed-phase high performance liquid chromatography. Journal of Chromatography.
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[8] Kidd, H. and D.R. James, 1991. The agrochemicals handbook, 3rd Edn. Royal Society of Chemistry
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[9] Ralph, I. and L. Ronald, 1976. Separation of rotenoids by high performance liquid chromatography. Journal
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[10] Zubairi S.I., M.R. Sarmidi and R.A. Aziz, 2014. Identification of bio-active constituents from Derris
elliptica liquid crude extract using vacuum liquid chromatography. Adv. Environ. Biol. 8(2): 437-440.
[11] Zubairi S.I., M.R. Sarmidi and R.A. Aziz, 2014. Precipitation of rotenoids resin extracted from Derris
elliptica roots by means of clarifying agents. Adv. Environ. Biol. 8(2): 441-444.
[12] AOAC Official Method 983.06, 2000. Rotenone in Pesticide Formulations. Liquid Chromatographic
Method. First Action 1983 and Final Action 1991. AOAC OFFICIAL METHOD OF ANALYSIS.
[13] Zubairi S.I., M.R. Sarmidi and R.A. Aziz, 2014. A study of rotenone from Derris roots of varies location,
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