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Cited by 15Insect Pest Control StrategiesPhytochemical compounds biological activitiesEssential Oils and Antimicrobial Activity

Simultaneous Extraction and Separation of Oil and Azadirachtin from Seeds and Leaves of Azadirachta indica using Binary Solvent Extraction

Open access
Subramanian, Sheela, Salleh, Aiza Syuhaniz, Bachmann, Robert Thomas, Hossain, Md. Sohrab·2019·Natural Product Sciences
DOI 10.20307/nps.2019.25.2.150
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Abstract

Conventional extraction of oil and azadirachtin, a botanical insecticide, from Azadirachta indica involves defatting the seeds and leaves using hexane followed by azadirachtin extraction with a polar solvent.In order to simplify the process while maintaining the yield we explored a binary extraction approach using Soxhlet extraction device and hexane and ethanol as non-polar and polar solvents at various ratios and extraction times.The highest oil and azadirachtin yields were obtained at 6 h extraction time using a 50:50 solvent mixture for both neem leaves (44.7 wt%, 720 mg Aza /kg leaves ) and seeds (53.5 wt%, 1045 mg Aza /kg seeds ), respectively.

AI snapshot

Written from the paper’s full text.

TL;DR

Binary solvent Soxhlet extraction with n-hexane:ethanol (50:50) at 6 h gave the highest simultaneous oil and azadirachtin yields from neem leaves and seeds. The study reports yields of 44.7 wt% oil and 720 mg azadirachtin/kg leaves, and 53.5 wt% oil and 1045 mg azadirachtin/kg seeds.

Key findings

  • For neem leaves, the highest oil and azadirachtin yields were 44.7 wt% and 720 mg/kg at a 50:50 n-hexane:ethanol ratio and 6 h extraction; for neem seeds, the highest were 53.5 wt% oil and 1045 mg/kg azadirachtin under the same 50:50 ratio and 6 h.
  • Single solvents gave lower yields than the 50:50 mixture: leaf oil yields were 11 ± 1 wt% lower with n-hexane and 24 ± 2 wt% lower with ethanol; seed oil yields were 7 ± 1 wt% lower with n-hexane and 17 ± 2 wt% lower with ethanol. Azadirachtin from leaves was 2.7–7.8 times lower with n-hexane and 1.9–3.7 times lower with ethanol; from seeds it was 3.0–5.5 times lower with n-hexane and 2.0–2.7 times lower with ethanol.
  • Oil yield increased linearly with extraction time (R² > 0.98 for leaves, R² > 0.99 for seeds), and azadirachtin concentration also increased linearly (R² > 0.98). Two-way ANOVA showed significant time × solvent-ratio interaction for azadirachtin in seeds and leaves and for leaf oil, but not for seed oil (F = 0.42).
  • The paper suggests neem seeds are the primary source for commercial azadirachtin production, based on higher azadirachtin yield from seeds than leaves.

Methods

  • Soxhlet extraction was performed with 10 g sample, 1:15 mass-to-solvent ratio, particle size 0.425–0.710 mm, and n-hexane:ethanol ratios of 100:0, 60:40, 50:50, 40:60, and 0:100; extraction times were 3, 4, 5, and 6 h, with experiments in triplicate.
  • Fresh neem leaves were collected from a 12-year-old tree in Mambau, Malaysia; neem seeds were procured from Tamil Nadu, India. Leaves and seeds were dried, ground, and sieved before extraction.
  • Azadirachtin was separated and quantified by HPLC using a C18 column (250 mm × 4.6 mm id × 5 μm), acetonitrile:water (72.5:27.5, v/v) mobile phase, 215 nm detection, 1 mL/min flow, and azadirachtin retention time of 15.85 min. Data were analyzed by two-way ANOVA.

Limitations

  • The paper does not state limitations explicitly.
  • Evident from the text: oil yield increased linearly with extraction time, suggesting maximum oil recovery may occur beyond the tested 6 h, so the tested range may not capture the maximum.
  • Evident from the text: seasonal and geographic variations are noted as factors that can affect plant composition and may account for deviations from literature; the study used one leaf source and one seed source.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.20307/nps.2019.25.2.150

Cites

13 of 13 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

1 of 17 cited sources checked · 0 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    For neem leaves, single solvent n-hexane and ethanol gave 11 ± 1 wt% and 24 ± 2 wt% lower oil yields, respectively, compared to the 50:50 binary solvent at 6 h extraction time.

    “The extraction using the single solvent n-hexane and ethanol resulted in 11 ± 1 wt% and 24 ± 2 wt% lower oil yields compared to the binary solvent ratio of 50:50 at extraction time of 6 h.”
    Quote in the paperSubstantial claim
  • Findingown data

    The concentration of azadirachtin in neem seed oil increased linearly with extraction time (R2 > 0.98).

    “The concentration of azadirachtin in neem seed oil increased linearly with extraction time (R 2 > 0.98) and reached comparatively high concentrations (Table 1).”
    Quote in the paperNot a substantial claim
  • Findingown data

    For neem seeds, the highest oil yield was 53.5 wt% at 6 h extraction time using a 50:50 n-hexane:ethanol solvent mixture.

    “The highest oil and azadirachtin yields were obtained at 6 h extraction time using a 50:50 solvent mixture for both neem leaves (44.7 wt%, 720 mg Aza /kg leaves ) and seeds (53.5 wt%, 1045 mg Aza /kg seeds ), respectively.”
    • Also stated in: § Results and Discussion · plain
      “The highest oil yield obtained was 53.5% using a binary solvent system of 50:50 followed by 60:40, 40:60, 100:0 and 0:100 (Fig. 2a)”
    Quote in the paperSubstantial claim
  • Findingown data

    For neem leaves, binary solvent had a higher affinity for neem oil and azadirachtin than single solvents at the studied solvent ratios and extraction times.

    “It was observed that binary solvent had a higher affinity for neem oil and azadirachtin from neem leaves at the studied solvent ratios and extraction times.”
    Quote in the paperSubstantial claim
  • Findingown data

    For neem leaves, azadirachtin yield was higher using ethanol as single solvent than using n-hexane as single solvent.

    “Conversely, the extracted azadirachtin yield from neem leaves was higher using ethanol as single solvent.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 14
  • May combine two claims. 10
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    A quality control was included using methanol injection at the beginning and after 5 samples.

    “A quality control was included using methanol injection at the beginning and after 5 samples.20”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [20] Carvalho, 2012, Braz J Chem Eng source

      The Material and Obtaining the Fatty Acids sections (page numbers unavailable) describe the reagents and chromatographic injections, but neither mentions methanol injections for quality control. Searches for methanol injection, quality-control blanks, and injections after five samples found no supporting passage in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    For neem seeds, azadirachtin concentration in n-hexane-extracted neem oil was 3.0 to 5.5 times lower than the maximum concentration obtained with the 50:50 n-hexane:ethanol solvent mixture, whereas pure ethanol extracted neem oil contained 2.0 to 2.7 times lower azadirachtin concentrations.

    “Azadirachtin concentration in n-hexane-extracted neem oil was 3.0 to 5.5 times lower than the maximum concentration obtained with the 50:50 n-hexane:ethanol solvent mixture, whereas pure ethanol extracted neem oil contained 2.0 to 2.7 lower azadirachtin concentrations.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    For neem leaves, the highest oil yield was 44.7 wt% at 6 h extraction time using a 50:50 n-hexane:ethanol solvent mixture.

    “The highest oil and azadirachtin yields were obtained at 6 h extraction time using a 50:50 solvent mixture for both neem leaves (44.7 wt%, 720 mg Aza /kg leaves ) and seeds (53.5 wt%, 1045 mg Aza /kg seeds ), respectively.”
    • Also stated in: § Results and Discussion · hedged
      “The highest oil and azadirachtin yields were about 45% and 720 mg/kg, respectively, at a solvent ratio of 50:50 for 6 h extraction time.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The ethanol-based oil yield from neem leaves is in excellent agreement with literature reporting a similar yield using the same extraction time and particle size range.

    “The ethanol-based oil yield is in excellent agreement with literature 11 whichreported a similar yield using same extraction time and particle size range (Table 1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Evbuomwan, 2015, Eur Int J Sci Technol

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    For neem seeds, the highest azadirachtin yields were 317 mg/kg for n-hexane (6 h), 448 mg/kg for ethanol (6 h), and 1045 mg/kg for 50:50 binary solvent mixture (6 h).

    “The highest yields of azadirachtin from neem seed were found to be 317 mg/ kg for n-hexane (6 h), 448 mg/kg for ethanol (6 h) and 1045 mg/kg for 50:50 binary solvent mixture (6 h), which falls within the range of 300 to 2500 mg/kg suggesting that the variability is caused by the extraction technology used, climate and geographic location.21”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Elteraifi, 2011, Int J Biol Chem Sci

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The oil yield after 6 h of n-hexane extraction was 5% greater than reported elsewhere.

    “The oil yield after 6 h of n-hexane extraction was 5% greater than reported elsewhere.2”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Adewoye, 2012, J Nat Sci Res

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    For neem leaves, azadirachtin yields in single solvent extraction using n-hexane and ethanol were 2.7 to 7.8 times and 1.9 to 3.7 times lower, respectively, than the maximum azadirachtin concentration at 50:50 binary solvent.

    “Azadirachtin yield obtained from neem leaves in single solvent extraction using n-hexane and ethanol were 2.7 to 7.8 times and 1.9 to 3.7 times lower than the maximum azadirachtin concentrations at binary solvent ratio of 50:50 (n-hexane:ethanol).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For neem seeds, the use of n-hexane as sole solvent (100:0) resulted in a 7 ± 1 wt% lower oil yield compared to 50:50, while ethanol (0:100) resulted in a 17 ± 2 wt% lower oil yield.

    “The use of n-hexane as the sole solvent (solvent ratio 100:0) resulted in a 7 ± 1 wt% lower oil yield compared to solvent ratio of 50:50, while ethanol yield was 17 ± 2 wt% lower, which, as far as single solvent extraction performance is concerned, confirms similar trends reported in literature (Table 1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Polar solvents such as ethanol and isopropanol can remove phospholipids from the plant cell wall, which improves oil permeability and facilitates faster extraction.

    “It has been reported that polar solvents such as ethanol and isopropanol can remove phospholipids from the plant cell wall which in turn improves oil permeability and thus facilitates a faster extraction.23”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Davoudpour, 2015, Ind Crop Prod source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Oil yield depends on moisture content, extraction time, size of particle and solvent:solid ratio.

    “Oil yield further depends on moisture content, extraction time, size of particle and solvent:solid ratio.13”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Ng, 2014, J Eng Sci Technol

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The extraction was carried out within 2 to 8 h with 6 h the most common extraction time.

    “The extraction was carried out within 2 to 8 h with 6 h the most common extraction time (Table 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    It is generally recommended that the R2 value should be ≥ 0.80 for a good fitted model.

    “It is generally recommended that the R 2 value should be ≥ 0.80 for a good fitted model.23”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Davoudpour, 2015, Ind Crop Prod source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Seasonal and geographic variations affect the composition of plant products.

    “Seasonal and geographic variations are also known to affect the composition of plant products 22 and may thus also account for the observed deviation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Sundaram, 1996, Environ Sci Health B; source

  • Classified as probably not a substantial claim.

    Premisecitation

    Polar solvents extract more azadirachtin than nonpolar solvent from n-hexane defatted neem seeds.

    “Another study investigated the effect of various solvents and found that polar solvents extract more azadirachtin than nonpolar solvent extracted from n-hexane defatted neem seeds suggesting solvent mixing maximizes azadirachtin yield 19 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Masson, 2007, J Chromatogr A source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    The majority of authors suggested a 1:15 solid to solvent ratio and preferred a particle size range of 0.425-0.71 mm.

    “The majority of authors suggested to use a 1:15 solid to solvent ratio and preferred a particle size range of 0.425 -0.71 mm.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Both solvent type and extraction time, as well as their interaction, are needed to generate yield for oil and azadirachtin.

    “Thus, both solvent types and extraction time is needed, as well as their interaction, to generate yield for oil and azadirachtin.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The solvent typically used for oil extraction is n-hexane due to its high stability, boiling point, non-polarity, low corrosiveness and high oil yield.

    “The solvent typically used for oil extraction is n-hexane due to its high stability, boiling point, non-polarity, low corrosiveness and high oil yield.2,3,12”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [2] Adewoye, 2012, J Nat Sci Res

      Not checked in the free run.

    • cites [3] Liauw, 2008, ARPN J Eng Appl Sci

      Not checked in the free run.

    • cites [12] Ambalkar, 2011, Int J Chem Anal Sci

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The variability in azadirachtin yield is caused by the extraction technology used, climate, and geographic location.

    “The highest yields of azadirachtin from neem seed were found to be 317 mg/ kg for n-hexane (6 h), 448 mg/kg for ethanol (6 h) and 1045 mg/kg for 50:50 binary solvent mixture (6 h), which falls within the range of 300 to 2500 mg/kg suggesting that the variability is caused by the extraction technology used, climate and geographic location.21”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Elteraifi, 2011, Int J Biol Chem Sci

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    A lower seed:solvent ratio is expected to result in a lower oil recovery.

    “However, these authors used a lower seed: solvent ratio which, everything else equal, is expected to result in a lower oil recovery (Table 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Experiments were carried out in triplicate.

    “Experiments were carried out in triplicate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The HPLC was calibrated with a 0.5 mg Sigma A7430 azadirachtin standard.

    “A 0.5 mg Sigma A7430 azadirachtin standard was used to calibrate the HPLC.”
    Quote in the paperNot a substantial claim
21 of 21

  • The paper relies on this without support.

    Sequential single solvent extraction increases heat stress to other valuable non-oil seed components such as proteins and carbohydrates, with detrimental consequences to their quality and economical value.

    “Sequential single solvent extraction also increases heat stress to other valuable non-oil seed components such as proteins and carbohydrates with detrimental consequences to their quality and economical value.”
1 of 1

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Claim of an interaction for oil yield conflicts with the ANOVA table

    Minor

    The text says the time-solvent interaction is needed for oil and azadirachtin yield, but Table 3 does not mark the interaction for neem-seed oil as significant (F = 0.42). The other interaction entries are marked significant.

  • Neem-leaf n-hexane oil-yield reduction does not match the stated yields

    Minor

    The stated 50:50 yield is 44.7 wt%, versus 41.0% for n-hexane: a difference of 3.7 percentage points, not 11 wt%. Even as a relative reduction, it is about 8.3%, outside the stated 11 ± 1%.

    Reported 11 · recomputed 3.7

  • Reported two-hour extraction is absent from the experimental time settings

    Minor

    The results describe an increase from 2 to 6 hours, but Table 2 lists the extraction times as 3, 4, 5 and 6 hours.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 10

  1. Adewoye, 2012, J Nat Sci Res, 2, 66
  2. Liauw, 2008, ARPN J Eng Appl Sci, 3, 49
  3. Hao, 2014, Biochim Biophys Acta, 1846, 247
  4. Biswas, 2002, Curr Sci, 82, 1336
  5. Evbuomwan, 2015, Eur Int J Sci Technol, 4, 1
  6. Ambalkar, 2011, Int J Chem Anal Sci, 2, 10
  7. Ng, 2014, J Eng Sci Technol, 8, 79
  8. Kovo, 2006, Leonardo Electronic J Practices Technologies, 8, 29
  9. Esparza-Díaz, 2010, Biopestic Int, 6, 45
  10. Elteraifi, 2011, Int J Biol Chem Sci, 5, 1063

Cited by

Sorted by citations

Related

Authors
Subramanian, S., Salleh, A. S., Bachmann, R. T., & Hossain, M. S.
Affiliations
  • University of Kuala Lumpur (MY)
  • Universiti Sains Malaysia (MY)
Corresponding author
Bachmann, Robert Thomas
ORCID
Venue
Natural Product Sciences
Publisher
The Korean Society of Pharmacognosy
Published
2019
Volume
25
Issue
2
Pages
150
ISSN
  • 1226-3907 (ISSN-L)
  • 2288-9027
DOI
10.20307/nps.2019.25.2.150
OpenAlex
W2963598145
Cited by
15
References
12
Citations by year
2026: 3 · 2025: 5 · 2024: 2 · 2023: 4 · 2022: 1
FWCI
0.17
Type
journal article
Language
en
Open access
Locations
Natural Product Sciences (publishedVersion)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Insect Pest Control Strategies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical compounds biological activities
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Essential Oils and Antimicrobial Activity
Keywords
Azadirachta indica, neem oil, azadirachtin, botanical insecticides, simultaneous extraction, neem seeds, neem leaves, Soxhlet extraction, extraction yield

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Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-26.

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